https://wiki.gi.science.ru.nl/api.php?action=feedcontributions&feedformat=atom&user=JelleGI - User contributions [en]2026-05-01T18:50:39ZUser contributionsMediaWiki 1.43.1https://wiki.gi.science.ru.nl/index.php?title=Nikon_TIRF_widefield&diff=244Nikon TIRF widefield2025-11-24T15:17:28Z<p>Jelle: /* Technical specifications */</p>
<hr />
<div>== Manuals and user guides ==<br />
[[TIRF alignment guide]]<br />
== Current status ==<br />
The current status of the microscope can be found [[Current microscope status#Light microscopes|here]] and on the [https://bookings.science.ru.nl/category/general-instrumentation-gi/gi-light-microscopy-image-analysis booking website].<br />
== Biosafety and access ==<br />
The TIRF is located in an ML-1 room. See the [[General Instrumentation Department#Biosafety|Biosafety]] section on rules regarding working in this room, and how to get access.<br />
<br />
== Technical specifications ==<br />
'''Light sources and filters:'''<br />
<br />
'''Epifluorescence mode''' with LED light sources (''non-TIRF)'': Excitations: 385, 475, 550, 621 nm. Emissions: Band pass emission filters for blue (ca. 450), green (ca. 510), red (ca. 600) and far-red (ca. 670).<br />
<br />
'''TIRF-mode''' with lasers: Excitations: 488 nm, 561 nm, 640 nm. Emission filters: None</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Current_microscope_status&diff=234Current microscope status2025-11-20T15:14:13Z<p>Jelle: </p>
<hr />
<div>==== Department closure during the Christmas holiday (update 14.11) ====<br />
<br />
* The Huygens building and therefor the GI department will be closed during the Christmas holiday from Monday 22 December up to and including January 2nd. <br />
** Only people with special card access can enter the GI department. Remember to use the overwork timer!<br />
** No support staff will be on site. <br />
** Access to liquid nitrogen is not guaranteed.<br />
* On January 5-6-7 the Huygens building will inaccessible due to maintenance work. No one will be allowed inside; this includes people with extra card access. <br />
<br />
=== Light microscopes ===<br />
{| class="wikitable"<br />
|+<br />
!Microscope<br />
!Status<br />
!Notes<br />
!Date<br />
|-<br />
|[[Leica SP8X|SP8X]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Leica SP8 Liachroic|SP8-Lia]]<br />
|OK<br />
|488 laser functional again<br />
|20.11.2025<br />
|-<br />
|[[Leica Thunder|Thunder]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Leica Timelapse|Timelapse]]<br />
|OK<br />
|New camera installed<br />
|25.09.2025<br />
|-<br />
|[[PhaseView light sheet|Lightsheet]]<br />
|<br />
|Microscope fully functional, use 4TB SSD drive to take images<br />
|20.11.2025<br />
|-<br />
|[[DM2500]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Nikon sora|SoRa]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Nikon TIRF widefield|TIRF]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|Spinoza server<br />
|OK<br />
|<br />
|<br />
|}<br />
<br />
=== Electron microscopes and sample prep equipment ===<br />
{| class="wikitable"<br />
|+<br />
!Machine<br />
!Status<br />
!Notes<br />
!Date<br />
|-<br />
|[[JEOL1400 TEM|JEOL1400]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[JEOL2100 TEM|JEOL2100]]<br />
|Broken<br />
|HT problems. Contacted JEOL.<br />
No date as of yet, but will not happen in November<br />
<br />
|12.11.2025<br />
14.11.2025<br />
|-<br />
|<br />
|<br />
|STEM detector broken - under repair<br />
EDX detector broken<br />
|aug 2024<br />
pre 2023<br />
|-<br />
|[[Cryo-holder, cryo-dock and cryo-pumping station|Cryo-dock/station/holder]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[HPM100]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[EMPACT|EMpact]]<br />
|Broken<br />
|Unsure if can be fixed<br />
|01.09.2025<br />
|-<br />
|[[Vitrobot]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[UC-E microtome]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|UC7/FC7 cryo-microtome<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[CCU-010]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|Edward306<br />
|Broken<br />
|Under repair<br />
|<br />
|-<br />
|BAF400<br />
|Broken<br />
|Under repair<br />
|<br />
|-<br />
|Vibratome<br />
|OK<br />
|<br />
|<br />
|-<br />
|CPD-020<br />
|In storage<br />
|<br />
|<br />
|-<br />
|JFD-030<br />
|OK<br />
|<br />
|<br />
|}</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora_tips_tricks&diff=221Nikon sora tips tricks2025-10-16T07:51:58Z<p>Jelle: </p>
<hr />
<div>'''Tips & Tricks'''<br />
<br />
- The function "'''Auto-Capture'''" captures an image and autosaves it to the specified folder (configured in Auto-Capture Settings).<br />
<br />
-</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora&diff=220Nikon sora2025-10-16T07:51:40Z<p>Jelle: </p>
<hr />
<div>== Manuals and user guides ==<br />
[[Nikon sora functions|What can it do?]]<br />
<br />
[[Nikon sora switch on|How to '''switch on''']]<br />
<br />
[[Nikon sora superresolution switch|How to '''switch to superresolution''']]<br />
<br />
[[Nikon sora lightpath|Dual camera imaging wavelengths]]<br />
<br />
[[Nikon sora tips tricks|Tips & Tricks]]<br />
<br />
== Current status ==<br />
The current status of the microscope can be found [[Current microscope status#Light microscopes|here]] and on the [https://bookings.science.ru.nl/category/general-instrumentation-gi/gi-light-microscopy-image-analysis booking website]. <br />
<br />
== Biosafety and access ==<br />
The SoRa is located in an ML-1 room. See the [[General Instrumentation Department#Biosafety|Biosafety]] section on rules regarding working in this room, and how to get access.<br />
<br />
== Technical specifications ==<br />
<br />
=== Available hardware ===<br />
<br />
* 20x air obj.<br />
* 40x/1.30 oil (working distance 200 um)<br />
* 100x/1.49 oil (w.d. 160 um)<br />
<br />
=== Superresolution components ===<br />
<br />
* SoRa spinning disc<br />
* Additional SoRa magnifiers 2.8x / 4.0x<br />
* 3D deconvolution software (in-line and parallel)<br />
<br />
=== Imaging lasers ===<br />
<br />
* 405 nm<br />
* 445 nm<br />
* 488 nm<br />
* 561 nm<br />
* 638 nm<br />
<br />
=== Emission filters ===<br />
<br />
* UV<br />
* Green<br />
* Yellow<br />
* Red<br />
* Far-red<br />
<br />
=== Dual color simultaneous imaging ===<br />
<br />
* blue/yellow (e.g. CFP / YFP)<br />
* green/red (e.g. GFP / RFP)<br />
<br />
=== Software ===<br />
<br />
* NIS-elements<br />
* Nikon batch-deconvolution<br />
<br />
=== Other ===<br />
<br />
* FURA (calcium sensor) imaging filters and light source<br />
* "Tokai Hit" Stage-top live-cell incubation system (CO2, temperature and humidity control)</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora_tips_tricks&diff=219Nikon sora tips tricks2025-10-16T07:51:06Z<p>Jelle: Created page with "Tips & Tricks - The function "'''Auto-Capture'''" captures an image and autosaves it to the specified folder (configured in Auto-Capture Settings). -"</p>
<hr />
<div>Tips & Tricks<br />
<br />
- The function "'''Auto-Capture'''" captures an image and autosaves it to the specified folder (configured in Auto-Capture Settings).<br />
<br />
-</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Current_microscope_status&diff=218Current microscope status2025-10-03T11:14:27Z<p>Jelle: </p>
<hr />
<div>=== Light microscopes ===<br />
{| class="wikitable"<br />
|+<br />
!Microscope<br />
!Status<br />
!Notes<br />
!Date<br />
|-<br />
|[[Leica SP8X|SP8X]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Leica SP8 Liachroic|SP8-Lia]]<br />
|OK<br />
|488 laser dodgy - don't turn it off!<br />
|27.08.2025<br />
|-<br />
|[[Leica Thunder|Thunder]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Leica Timelapse|Timelapse]]<br />
|OK<br />
|New camera installed<br />
|25.09.2025<br />
|-<br />
|[[PhaseView light sheet|Lightsheet]]<br />
|<br />
|561 laser weak+ software out of date<br />
Broken parts are sent for repair (PhaseView corp., Paris, France)<br />
|03.10.2025<br />
|-<br />
|[[DM2500]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Nikon sora|SoRa]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Nikon TIRF widefield|TIRF]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|Spinoza server<br />
|OK<br />
|<br />
|<br />
|}<br />
<br />
=== Electron microscopes and sample prep equipment ===<br />
{| class="wikitable"<br />
|+<br />
!Machine<br />
!Status<br />
!Notes<br />
!Date<br />
|-<br />
|[[JEOL1400 TEM|JEOL1400]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[JEOL2100 TEM|JEOL2100]]<br />
|OK<br />
|STEM detector broken - under repair<br />
EDX detector broken<br />
|aug 2024<br />
pre 2023<br />
|-<br />
|[[Cryo-holder, cryo-dock and cryo-pumping station|Cryo-dock/station/holder]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[HPM100]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[EMPACT|EMpact]]<br />
|Broken<br />
|Unsure if can be fixed<br />
|01.09.2025<br />
|-<br />
|[[Vitrobot]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[UC-E microtome]]<br />
|Broken<br />
|Under repair - contact Hetty if you need it soon<br />
|01.09.2025<br />
|-<br />
|UC7/FC7 cryo-microtome<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[CCU-010]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|Edward306<br />
|Broken<br />
|Under repair<br />
|<br />
|-<br />
|BAF400<br />
|Broken<br />
|Under repair<br />
|<br />
|-<br />
|Vibratome<br />
|OK<br />
|<br />
|<br />
|-<br />
|<br />
|<br />
|<br />
|<br />
|}</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Current_microscope_status&diff=217Current microscope status2025-10-03T11:14:14Z<p>Jelle: </p>
<hr />
<div>=== Light microscopes ===<br />
{| class="wikitable"<br />
|+<br />
!Microscope<br />
!Status<br />
!Notes<br />
!Date<br />
|-<br />
|[[Leica SP8X|SP8X]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Leica SP8 Liachroic|SP8-Lia]]<br />
|OK<br />
|488 laser dodgy - don't turn it off!<br />
|27.08.2025<br />
|-<br />
|[[Leica Thunder|Thunder]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Leica Timelapse|Timelapse]]<br />
|OK<br />
|New camera installed<br />
|25.09.2025<br />
|-<br />
|[[PhaseView light sheet|Lightsheet]]<br />
|<br />
|561 laser weak+ software out of date<br />
Broken parts are sent for repair (PhaseView corp., Paris, France)<br />
|?<br />
|-<br />
|[[DM2500]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Nikon sora|SoRa]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Nikon TIRF widefield|TIRF]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|Spinoza server<br />
|OK<br />
|<br />
|<br />
|}<br />
<br />
=== Electron microscopes and sample prep equipment ===<br />
{| class="wikitable"<br />
|+<br />
!Machine<br />
!Status<br />
!Notes<br />
!Date<br />
|-<br />
|[[JEOL1400 TEM|JEOL1400]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[JEOL2100 TEM|JEOL2100]]<br />
|OK<br />
|STEM detector broken - under repair<br />
EDX detector broken<br />
|aug 2024<br />
pre 2023<br />
|-<br />
|[[Cryo-holder, cryo-dock and cryo-pumping station|Cryo-dock/station/holder]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[HPM100]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[EMPACT|EMpact]]<br />
|Broken<br />
|Unsure if can be fixed<br />
|01.09.2025<br />
|-<br />
|[[Vitrobot]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[UC-E microtome]]<br />
|Broken<br />
|Under repair - contact Hetty if you need it soon<br />
|01.09.2025<br />
|-<br />
|UC7/FC7 cryo-microtome<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[CCU-010]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|Edward306<br />
|Broken<br />
|Under repair<br />
|<br />
|-<br />
|BAF400<br />
|Broken<br />
|Under repair<br />
|<br />
|-<br />
|Vibratome<br />
|OK<br />
|<br />
|<br />
|-<br />
|<br />
|<br />
|<br />
|<br />
|}</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Current_microscope_status&diff=216Current microscope status2025-10-03T11:13:23Z<p>Jelle: </p>
<hr />
<div>=== Light microscopes ===<br />
{| class="wikitable"<br />
|+<br />
!Microscope<br />
!Status<br />
!Notes<br />
!Date<br />
|-<br />
|[[Leica SP8X|SP8X]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Leica SP8 Liachroic|SP8-Lia]]<br />
|OK<br />
|488 laser dodgy - don't turn it off!<br />
|27.08.2025<br />
|-<br />
|[[Leica Thunder|Thunder]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Leica Timelapse|Timelapse]]<br />
|OK<br />
|New camera installed<br />
|25.09.2025<br />
|-<br />
|[[PhaseView light sheet|Lightsheet]]<br />
|<br />
|561 laser weak+ software out of date<br />
Broken microscope parts are sent for repair (PhaseView corp., Paris, France)<br />
|?<br />
|-<br />
|[[DM2500]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Nikon sora|SoRa]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Nikon TIRF widefield|TIRF]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|Spinoza server<br />
|OK<br />
|<br />
|<br />
|}<br />
<br />
=== Electron microscopes and sample prep equipment ===<br />
{| class="wikitable"<br />
|+<br />
!Machine<br />
!Status<br />
!Notes<br />
!Date<br />
|-<br />
|[[JEOL1400 TEM|JEOL1400]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[JEOL2100 TEM|JEOL2100]]<br />
|OK<br />
|STEM detector broken - under repair<br />
EDX detector broken<br />
|aug 2024<br />
pre 2023<br />
|-<br />
|[[Cryo-holder, cryo-dock and cryo-pumping station|Cryo-dock/station/holder]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[HPM100]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[EMPACT|EMpact]]<br />
|Broken<br />
|Unsure if can be fixed<br />
|01.09.2025<br />
|-<br />
|[[Vitrobot]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[UC-E microtome]]<br />
|Broken<br />
|Under repair - contact Hetty if you need it soon<br />
|01.09.2025<br />
|-<br />
|UC7/FC7 cryo-microtome<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[CCU-010]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|Edward306<br />
|Broken<br />
|Under repair<br />
|<br />
|-<br />
|BAF400<br />
|Broken<br />
|Under repair<br />
|<br />
|-<br />
|Vibratome<br />
|OK<br />
|<br />
|<br />
|-<br />
|<br />
|<br />
|<br />
|<br />
|}</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Leica_SP8X&diff=215Leica SP8X2025-09-30T10:55:05Z<p>Jelle: </p>
<hr />
<div>The SP8x confocal laser scanning microscope (Leica-microsystems) is mounted on a fully motorized DMi8 microscope on which x, y, z, t; lambda (wavelength), and sequential scans can be acquired at multiple positions and in z-stack. There are functions for stitching, and digital “best focus” determination also in z-stacks. Deconvolution can be done on the attached CUDA PC equipped with the LAS X module "Lightning". Live-cell imaging can be performed using a closed lid that controls CO2 and relative humidity levels, and using the big incubator box that controls temperature and airflow. <br />
<br />
== Manuals and user guides ==<br />
<br />
== Current status ==<br />
The current status of the microscope can be found [[Current microscope status#Light microscopes|here]] and on the [https://bookings.science.ru.nl/category/general-instrumentation-gi/gi-light-microscopy-image-analysis booking website].<br />
<br />
== Biosafety and access ==<br />
The SP8-Lia is located in an AP-1 room. See the [[General Instrumentation Department#Biosafety|Biosafety]] section on rules regarding working in this room.<br />
<br />
== Technical specifications ==<br />
<br />
=== Objectives ===<br />
<br />
* 10x / 0.40 Air (FWD = 2.2 mm)<br />
* 40x / 0.60 Air (3.3 mm long distance)<br />
* 40x / 1.10 Water (0.62 mm) - motorCorrection<br />
* 63x / 1.40 Oil (0.14 mm)<br />
* 100x / 1.40 Oil (0.13 mm)<br />
<br />
===Lasers===<br />
* 405 nm diode laser + pulsed White Light Laser (WLL), up to 8 tunable excitation lines from 470-670nm can be applied simultaneously<br />
<br />
===Detectors and camera===<br />
* 2 GaAsP PMTs<br />
* 2 HyDs (high sensitivity, linear photon counting and time-gating)<br />
* 1 normal transmitted-light PMT<br />
<br />
In addition, the DMi8 microscope can be utilized for wide-field bright field and fluorescence observation. Images can be acquired with a monochrome DFC365FX camera.<br />
<br />
===Filter cubes===<br />
* DAPI (LP425)<br />
* FITC (LP515)<br />
* Rhod (LP590)<br />
* Y5 (BP 662-738)</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Current_microscope_status&diff=154Current microscope status2025-09-26T09:20:42Z<p>Jelle: </p>
<hr />
<div>=== Light microscopes ===<br />
{| class="wikitable"<br />
|+<br />
!Microscope<br />
!Status<br />
!Notes<br />
!Date<br />
|-<br />
|SP8X<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Leica SP8 Liachroic|SP8-Lia]]<br />
|OK<br />
|488 laser dodgy - don't turn it off!<br />
|27.08.2025<br />
|-<br />
|Thunder<br />
|OK<br />
|<br />
|<br />
|-<br />
|Timelapse<br />
|OK<br />
|New camera installed<br />
|25.09.2025<br />
|-<br />
|Lightsheet<br />
|<br />
|561 laser weak<br />
Software out of date<br />
<br />
service visit planned 03.10.2025<br />
|?<br />
|-<br />
|DM2500<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Nikon sora|SoRa]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Nikon TIRF widefield|TIRF]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|Spinoza server<br />
|OK<br />
|<br />
|<br />
|}<br />
<br />
=== Electron microscopes and sample prep equipment ===<br />
{| class="wikitable"<br />
|+<br />
!Machine<br />
!Status<br />
!Notes<br />
!Date<br />
|-<br />
|[[JEOL1400 TEM|JEOL1400]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|JEOL2100<br />
|OK<br />
|STEM detector broken - under repair<br />
EDX detector broken<br />
|aug 2024<br />
pre 2023<br />
|-<br />
|Cryo-dock/station/holder<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[HPM100]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[EMPACT|EMpact]]<br />
|Broken<br />
|Unsure if can be fixed<br />
|01.09.2025<br />
|-<br />
|Vitrobot<br />
|OK<br />
|<br />
|<br />
|-<br />
|UC-E<br />
|Broken<br />
|Under repair - contact Hetty if you need it soon<br />
|01.09.2025<br />
|-<br />
|UC7/FC7 cryo-microtome<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[CCU-010]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|Edward306<br />
|Broken<br />
|Under repair<br />
|<br />
|-<br />
|BAF400<br />
|Broken<br />
|Under repair<br />
|<br />
|-<br />
|Vibratome<br />
|OK<br />
|<br />
|<br />
|-<br />
|<br />
|<br />
|<br />
|<br />
|}</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Current_microscope_status&diff=153Current microscope status2025-09-26T09:20:32Z<p>Jelle: </p>
<hr />
<div>=== Light microscopes ===<br />
{| class="wikitable"<br />
|+<br />
!Microscope<br />
!Status<br />
!Notes<br />
!Date<br />
|-<br />
|SP8X<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Leica SP8 Liachroic|SP8-Lia]]<br />
|OK<br />
|488 laser dodgy - don't turn it off!<br />
|27.08.2025<br />
|-<br />
|Thunder<br />
|OK<br />
|<br />
|<br />
|-<br />
|Timelapse<br />
|OK<br />
|New camera installed<br />
|25.09.2025<br />
|-<br />
|Lightsheet<br />
|<br />
|561 laser weak<br />
Software out of date<br />
service visit planned 03.10.2025<br />
|?<br />
|-<br />
|DM2500<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Nikon sora|SoRa]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[Nikon TIRF widefield|TIRF]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|Spinoza server<br />
|OK<br />
|<br />
|<br />
|}<br />
<br />
=== Electron microscopes and sample prep equipment ===<br />
{| class="wikitable"<br />
|+<br />
!Machine<br />
!Status<br />
!Notes<br />
!Date<br />
|-<br />
|[[JEOL1400 TEM|JEOL1400]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|JEOL2100<br />
|OK<br />
|STEM detector broken - under repair<br />
EDX detector broken<br />
|aug 2024<br />
pre 2023<br />
|-<br />
|Cryo-dock/station/holder<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[HPM100]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[EMPACT|EMpact]]<br />
|Broken<br />
|Unsure if can be fixed<br />
|01.09.2025<br />
|-<br />
|Vitrobot<br />
|OK<br />
|<br />
|<br />
|-<br />
|UC-E<br />
|Broken<br />
|Under repair - contact Hetty if you need it soon<br />
|01.09.2025<br />
|-<br />
|UC7/FC7 cryo-microtome<br />
|OK<br />
|<br />
|<br />
|-<br />
|[[CCU-010]]<br />
|OK<br />
|<br />
|<br />
|-<br />
|Edward306<br />
|Broken<br />
|Under repair<br />
|<br />
|-<br />
|BAF400<br />
|Broken<br />
|Under repair<br />
|<br />
|-<br />
|Vibratome<br />
|OK<br />
|<br />
|<br />
|-<br />
|<br />
|<br />
|<br />
|<br />
|}</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Leica_SP8_Liachroic_intro&diff=109Leica SP8 Liachroic intro2025-09-23T10:02:50Z<p>Jelle: </p>
<hr />
<div>'''Introduction'''<br />
<br />
# Welcome, outline of what the introduction will be (takes a few hours)<br />
# The General Instrumentation department where we are<br />
# The room and the general layout (sink, microscope, cupboard, chairs)<br />
# The microscope conceptually (Confocal Laser Scanning Microscope)<br />
## '''Confocal''' = observing only a small horizontal layer of the sample, not seeing blurry higher or lower objects.<br />
## '''Laser''' = specific wavelengths, focused spot, to only see specifically what we want<br />
## '''Scanning''' = doesn't take a picture in one shot, but scans every pixel by pixel until the whole picture of a square field of view is built.<br />
# The microscope physically (components, roughly what parts do we see? -> microscope, scanner (left, attached to microscope on air table), laser box (left next to air table))<br />
<br />
'''Switch-on'''<br />
<br />
# Quick inspection (everything looks ok visually?)<br />
# Switch-on procedure of components (buttons in numbered order)<br />
# Log-in in Windows (use yours, user will get an account) and software starting (LAS X)<br />
<br />
'''Stage and objective movement controls''' (XY stage joystick, Z objectives, Switch Objectives on the touch screen - start with the 10X lens. You normally start low, then go higher)<br />
<br />
'''Sample placement''' (sample types, coverslip towards the bottom, adjusting holder, put demo or user sample there, don't clamp it but let it rest (otherwise breaks when objective hits))<br />
<br />
'''Brightfield''' (where does light come from, what do we expect to see)<br />
<br />
'''Focusing''' (sample finding, and then moving around, warning: hitting lens to sample possible)<br />
<br />
'''Epifluorescence''' (see visually light shining, see returning light, discuss different filters, user responsible for knowing their dyes and wavelengths, Searchlight spectra viewer. This method is used to see where is the signal, is there any signal, do I already see something suitable for confocal imaging?)<br />
<br />
'''Confocal scanning''' (gonna be a big part, mostly on computer now, switch in mood/user to focus on screen not on microscope - instructor sits on the far-right side of the desk to control the mouse, attendees sit right in front of the screen to see everything properly)<br />
<br />
- Launch the software LAS X which controls the microscope, and choose the right startup settings (microscope = DMi8, settings = standard) and wait for it to appear completely. The software is going to ignore any wavelength/imaging modality (BF, EPI) choices you have made using the touch screen of the microscope and do confocal anyway.<br />
<br />
'''- Overview of software areas:'''<br />
<br />
# We start in "Acquire" (top) and will be spending most time there<br />
# The big vertical line in the middle divides the view into settings (left) and image display (right)<br />
# Making any adjustments to settings in the image display (right) is not going to change any raw data values or any microscope settings - just how the image looks on the screen<br />
# Making adjustments on the left will change actual microscope settings, such as wavelengths used, scanning behavior etc.<br />
# At the bottom, there are the buttons that make the microscope actually do something (Live, Capture Image, Start - etc.) - to prevent confusion when clicking "open laser shutter" in the software, or any other buttons.<br />
# We make 1 small excursion to "Configuration" (left of "Acquire") and then "Laser Config" in order to switch on the lasers we know we will need.<br />
<br />
'''- Laser setup''' <br />
<br />
# choose the right wavelength<br />
# give it a tiny bit of power (philosophy: start low, go higher if needed - instead of bleaching everything straight away)<br />
# click round button under the laser to open the shutter<br />
# Confirm yellow line appears in the schematic light path below -> laser ready.<br />
<br />
'''- Detector setup'''<br />
<br />
# Acknowledge we have a few detectors available, one is switched on.<br />
# Prefer to always choose HyD (hybrid detectors), they are better. Typically they are in standard mode with 100 gain (digital gain) and you can use them like that. You can destroy the detector by setting its detection window to include the current laser wavelength used (dotted line in detector area spectrum reminds you which lasers are used).<br />
# PMT detectors are less sensitive = good for strong signals (usually you have weak signals), to use one you have to give it some gain (couple hundred Volts, until image looks good to you)<br />
# Move the detection window and resize it to capture the desired fluorescence emission wavelengths. Avoid including the laser wavelength at all cost!! This will visualise the laser, which we do not want to see - and it can damage the detector.<br />
<br />
'''- First image'''<br />
<br />
# Click "Capture Image"<br />
# Probably, nothing will show up!<br />
# Is there something there, but just weak? -> Set the Image Display (right) to "auto-scale" (button called "M" because it starts on Manual scale) -> have a look if something shows up.<br />
# Is there nothing there anyway, maybe you need more laser -> Increase laser power to 20-30%, capture another image -> have a look if something shows up.<br />
# Is there still nothing, maybe the focus is slightly wrong (we're in confocal mode after all...) -> use the Z-adjustment knob to focus up and down (either on the microscope, on the joystick, or on the USB panel) -> have a look if something shows up, now it really should.<br />
# If there's still nothing, visually check if the sample is still alright, and then use Brightfield or Epifluorescence through the binoculars to find focus again. Then try confocal again, using the software.<br />
# If there's still nothing, there is a problem with the microscope or the sample. Change samples to a known working sample - a piece of paper responds to 405 nm, a stroke of Staedler Yellow Highlighter responds to 488 nm.<br />
<br />
'''- Evaluating the first image, boundary conditions'''<br />
<br />
# It could be too weak (barely jumping out of the noise), so we need more laser until it's visible (improving the scan will help too, but this comes later) at the cost of possibly bleaching<br />
# It could be too strong (huge areas look intense, and same intensity - mouse-over the pixel and see the maximum value 255 show up under the picture = too bright!) you want to avoid saturation.<br />
<br />
'''- Optimizing the scan (better picture, takes longer, make it as good as you need - and not better. Every improvement you make can be compared to the previous situation by flipping through the last couple of acquired images)'''<br />
<br />
# This part is about losing acquisition speed but gaining image quality.<br />
# There are three main settings that most influence the image quality, which you can use to maximize the details and signals that the current objective is able to see. (if not enough, you need a higher mag. objective) as follows:<br />
# '''Format''' (number of pixels that are scanned -> note that the square area of the sample that you are scanning does not change in size, just gets divided into smaller steps i.e. more measurement points i.e. pixels). This influences the resolution of the image (lower = worse). Use the "Optimize" button on the left of Format to always get the right number of pixels to describe the details that are available through this objective.<br />
# '''Speed''' (lower values = slower, takes longer but results in higher signal-to-noise). This means the laser spends more time on each pixel, and thus illuminates that place for longer, and thus gets more signals if there are signals. Noise always stays the same.<br />
# '''Averaging''' (higher values = longer, also more signal-to-noise). This means one horizontal line will be acquired that number of times and the values averaged. Noise randomly appears everywhere. Signal always appears, on every scan consistently, when you look at a location that has signal.<br />
<br />
'''- Adding a second channel and using Sequential Scanning'''<br />
<br />
# Set the settings to "bad" again (512x512 pixels, 400 speed, no averaging) to save some time.<br />
# Set up another laser (e.g. 561, if you used 488) to activate the other fluorophores<br />
# Set up another detector (e.g. HyD3 if you used HyD1) to detect the other fluorophores' signals<br />
# Do another "Capture Image" and make optimizations where necessary (use the Evaluating the second image just like in the first image section above).<br />
# '''Sequential Scanning''': both molecule populations and both detectors are active at the same time, so bleedthrough might happen.<br />
# Switch on "SEQ" at the top left, a sequential scanning menu will appear.<br />
# Add another sequence using "+" and switch off one of the detectors in sequence 2<br />
# Flip back and forth between seq 1 and 2 so that you can see the difference in settings.<br />
# Now set up sequence 1 for one imaging channel, and sequence 2 for the other: Lasers should be "switched off" by putting their power on 0 where necessary, in each sequence.<br />
<br />
'''- Switching to a higher magnification lens (use navigator to register 1 pic, switch to immersion lens, go back to original location).'''<br />
<br />
# Higher magnification does not automatically mean higher detail on a confocal scanning microscope.<br />
# Higher NA does mean higher detail on a confocal. Provided that the scan settings are also optimized (just done that, can repeat it).<br />
<br />
'''- Z-stack''' (look at the menu: set start height, go to a different focus and set end height, and that's it - would always recommend to leave the rest of the Z-stack settings as-is for the introduction)<br />
<br />
'''- Time course''' (switch modes using dropdown menu, then set (1) interval (none, or amount of time) and (2) duration (unlimited, or amount of time, or amount of pictures)<br />
<br />
'''- Tilescan''' (Use the Navigator for this, do one small one, several of them, focus map, stitching)<br />
<br />
# Make sure some objects are in the view in normal viewer<br />
# Switch to '''Navigator''' and do "Spiral"<br />
# After an area is revealed click "'''Stop'''"<br />
# Use a '''drawing tool''' (bottom, e.g. the rectangle) to designate a small tilescan area<br />
# '''Execute''' ("Start") the tilescan<br />
# In the left menu "Stage", confirm that "Merge images during acquisition" is on, and "blend" is on "Smooth" <br />
# Inspect the result, be skeptical of overlap areas, they should be stitched well<br />
<br />
<br />
<br />
<br />
'''Rounding off'''<br />
<br />
- Saving data (save the projects as LIF files locally, then before ending your session move the raw data to a safe location e.g. through SURF or network drive, our policy is that the computer is not a safe storage space and we can't count on it)<br />
<br />
- Saving settings (sequential scan settings in the sequential scan menu, single settings above the laser set-up area, or you can reload settings from an image in a LIF file: right-click on one and "apply".<br />
<br />
- Closing software (is someone coming after us? do we need to keep the whole system on just logging out, or switch the whole system off?)<br />
<br />
- Cleaning oil objective, then put back at 10X<br />
<br />
- Shutting down microscope (if necessary, otherwise just logging out)<br />
<br />
- Leaving room and system tidy for next user!<br />
<br />
<br />
'''Optional functions'''<br />
<br />
- Autofocus<br />
<br />
- Emission wavelength scanning (check whether the signal behaves like the fluorophore should)<br />
<br />
- Closing the pinhole (more resolution, lower signal)<br />
<br />
- Changing Z-stack settings like step size (speed thigns up, but you may skip layers and thus lose information)</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora_lightpath&diff=108Nikon sora lightpath2025-09-05T09:57:43Z<p>Jelle: </p>
<hr />
<div>== Dual camera imaging situation and choices: ==<br />
[[File:Dual_cam_light_path.png|836x836px]]</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora&diff=107Nikon sora2025-09-05T09:57:33Z<p>Jelle: </p>
<hr />
<div>[[Nikon sora functions|What can it do?]]<br />
<br />
[[Nikon sora switch on|How to '''switch on''']]<br />
<br />
[[Nikon sora superresolution switch|How to '''switch to superresolution''']]<br />
<br />
[[Nikon sora lightpath|Dual camera imaging wavelengths]]</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora&diff=106Nikon sora2025-09-05T09:57:25Z<p>Jelle: added dual cam lightpath</p>
<hr />
<div>[[Nikon sora functions|What can it do?]]<br />
<br />
[[Nikon sora switch on|How to '''switch on''']]<br />
<br />
[[Nikon sora superresolution switch|How to '''switch to superresolution''']]<br />
<br />
[[Nikon sora lightpath|Dual camera simultaneous imaging wavelengths]]</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora_lightpath&diff=105Nikon sora lightpath2025-09-05T09:55:53Z<p>Jelle: </p>
<hr />
<div>'''Dual camera imaging situation and choices:'''<br />
<br />
[[File:Dual_cam_light_path.png|836x836px]]</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora_lightpath&diff=104Nikon sora lightpath2025-09-05T09:53:47Z<p>Jelle: Created page with "thumb"</p>
<hr />
<div>[[File:Dual cam light path.png|thumb]]</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=File:Dual_cam_light_path.png&diff=103File:Dual cam light path.png2025-09-05T09:53:42Z<p>Jelle: </p>
<hr />
<div>Nikon sora dual camera light path choices</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Leica_SP8_Liachroic_intro&diff=101Leica SP8 Liachroic intro2025-08-14T12:20:54Z<p>Jelle: additions</p>
<hr />
<div>'''Introduction'''<br />
<br />
# Welcome, outline of the introduction<br />
# The General Instrumentation department conceptually and physically<br />
# The room and the general layout<br />
# The microscope conceptually (functions)<br />
# The microscope physically (components, roughly)<br />
<br />
'''Switch-on'''<br />
<br />
# Quick inspection (everything looks ok visually?)<br />
# Switch-on procedure of components (buttons numbered order)<br />
# Log-in in Windows (user will get an account) and software starting<br />
<br />
'''Stage and objective movement controls''' (XY stage, Z objectives, Switch Objectives on the touch screen - start with the 10X lens. You normally start low, then go higher)<br />
<br />
'''Sample placement''' (sample types, coverslip towards the bottom, adjusting holder, put demo or user sample there, don't clamp it but let it rest (otherwise breaks when objective hits))<br />
<br />
'''Brightfield''' (where does light come from, what do we expect to see)<br />
<br />
'''Focusing''' (sample finding, and then moving around, warning: hitting lens to sample possible)<br />
<br />
'''Epifluorescence''' (see visually light shining, see returning light, discuss different filters, user responsible for knowing their dyes and wavelengths, Searchlight spectra viewer. This method is used to see where is the signal, is there any signal, do I already see something suitable for confocal imaging?)<br />
<br />
'''Confocal scanning''' (gonna be a big part, mostly on computer now, switch in mood/user to focus on screen not on microscope - instructor sits on the far-right side of the desk to control the mouse, attendees sit right in front of the screen to see everything properly)<br />
<br />
- Launch the software LAS X which controls the microscope, and choose the right startup settings (microscope = DMi8, settings = standard) and wait for it to appear completely. The software is going to ignore any wavelength/imaging modality (BF, EPI) choices you have made using the touch screen of the microscope and do confocal anyway.<br />
<br />
- Overview of software areas:<br />
<br />
# We start in "Acquire" (top) and will be spending most time there<br />
# The big vertical line in the middle divides the view into settings (left) and image display (right)<br />
# Making any adjustments to settings in the image display (right) is not going to change any raw data values or any microscope settings - just how the image looks on the screen<br />
# Making adjustments on the left will change actual microscope settings, such as wavelengths used, scanning behavior etc.<br />
# At the bottom, there are the buttons that make the microscope actually do something (Live, Capture Image, Start - etc.) - to prevent confusion when clicking "open laser shutter" in the software, or any other buttons.<br />
# We make 1 small excursion to "Configuration" (left of "Acquire") and then "Laser Config" in order to switch on the lasers we know we will need.<br />
<br />
- Laser setup <br />
<br />
# choose the right wavelength<br />
# give it a tiny bit of power (philosophy: start low, go higher if needed - instead of bleaching everything straight away)<br />
# click round button under the laser to open the shutter<br />
# Confirm yellow line appears in the schematic light path below -> laser ready.<br />
<br />
- Detector setup<br />
<br />
# Acknowledge we have a few detectors available, one is switched on.<br />
# Prefer to always choose HyD (hybrid detectors), they are better. Typically they are in standard mode with 100 gain (digital gain) and you can use them like that. You can destroy the detector by setting its detection window to include the current laser wavelength used (dotted line in detector area spectrum reminds you which lasers are used).<br />
# PMT detectors are less sensitive = good for strong signals (usually you have weak signals), to use one you have to give it some gain (couple hundred Volts, until image looks good to you)<br />
# Move the detection window and resize it to capture the desired fluorescence emission wavelengths. Avoid including the laser wavelength at all cost!! This will visualise the laser, which we do not want to see - and it can damage the detector.<br />
<br />
- First image<br />
<br />
# Click "Capture Image"<br />
# Probably, nothing will show up!<br />
# Is there something there, but just weak? -> Set the Image Display (right) to "auto-scale" (button called "M" because it starts on Manual scale) -> have a look if something shows up.<br />
# Is there nothing there anyway, maybe you need more laser -> Increase laser power to 20-30%, capture another image -> have a look if something shows up.<br />
# Is there still nothing, maybe the focus is slightly wrong (we're in confocal mode after all...) -> use the Z-adjustment knob to focus up and down (either on the microscope, on the joystick, or on the USB panel) -> have a look if something shows up, now it really should.<br />
# If there's still nothing, visually check if the sample is still alright, and then use Brightfield or Epifluorescence through the binoculars to find focus again. Then try confocal again, using the software.<br />
# If there's still nothing, there is a problem with the microscope or the sample. Change samples to a known working sample - a piece of paper responds to 405 nm, a stroke of Staedler Yellow Highlighter responds to 488 nm.<br />
<br />
- Evaluating the first image, boundary conditions<br />
<br />
# It could be too weak (barely jumping out of the noise), so we need more laser until it's visible (improving the scan will help too, but this comes later) at the cost of possibly bleaching<br />
# It could be too strong (huge areas look intense, and same intensity - mouse-over the pixel and see the maximum value 255 show up under the picture = too bright!) you want to avoid saturation.<br />
<br />
- Optimizing the scan (better picture, takes longer, make it as good as you need - and not better. Every improvement you make can be compared to the previous situation by flipping through the last couple of acquired images)<br />
<br />
# There are three main settings that most influence the image quality, which you can use to maximize the details and signals that the current objective is able to see. (if not enough, you need a higher mag. objective)<br />
# Format (number of pixels that are scanned -> note that the square area of the sample that you are scanning does not change in size, just gets divided into smaller steps i.e. more measurement points i.e. pixels). This influences the resolution of the image (lower = worse). Use the "Optimize" button on the left of Format to always get the right number of pixels to describe the details that are available through this objective.<br />
# Speed (lower values = slower, takes longer but results in higher signal-to-noise). This means the laser spends more time on each pixel, and thus illuminates that place for longer, and thus gets more signals if there are signals. Noise always stays the same.<br />
<br />
- Adding a second channel<br />
<br />
# Set up another laser (e.g. 561, if you used 488) to activate the other fluorophores<br />
# Set up another detector (e.g. HyD3 if you used HyD1) to detect the other fluorophores' signals<br />
# Do another "Capture Image" and make optimizations where necessary (use the Evaluating the first image section above)<br />
<br />
- Switching to a higher magnification lens (use navigator to register 1 pic, switch to immersion lens, go back to original location).<br />
<br />
# Higher magnification does not automatically mean higher detail on a confocal scanning microscope.<br />
# Higher NA does mean higher detail on a confocal. Provided that the scan settings are also optimized (just done that, can repeat it).<br />
<br />
- Z-stack (set start, end, use optimized)<br />
<br />
- Time course (switch modes using dropdown menu, then set (1) interval (none, or amount of time) and (2) duration (unlimited, or amount of time, or amount of pictures)<br />
<br />
- Tilescan (one small one, several of them, focus map, stitching)<br />
<br />
<br />
<br />
'''Rounding off'''<br />
<br />
- Saving data<br />
<br />
- Saving settings<br />
<br />
- Closing software<br />
<br />
- Shutting down microscope (and cleaning oil objective, then put back at 10X)<br />
<br />
- Leaving room and system tidy for next user<br />
<br />
<br />
Optional functions<br />
<br />
- Autofocus<br />
<br />
- Emission wavelength scanning (check whether the signal behaves like the fluorophore should)<br />
<br />
- Closing the pinhole (more resolution, lower signal)</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Leica_SP8_Liachroic&diff=98Leica SP8 Liachroic2025-08-06T16:36:47Z<p>Jelle: added link</p>
<hr />
<div>[[Leica SP8 Liachroic intro|Introduction protocol]]</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Leica_SP8_Liachroic_intro&diff=97Leica SP8 Liachroic intro2025-08-06T16:35:53Z<p>Jelle: drafted most segments and started adding details</p>
<hr />
<div>'''Introduction'''<br />
<br />
# Welcome, outline of the introduction<br />
# The General Instrumentation department conceptually and physically<br />
# The room and the general layout<br />
# The microscope conceptually (functions)<br />
# The microscope physically (components, roughly)<br />
<br />
'''Switch-on'''<br />
<br />
# Quick inspection (everything looks ok visually?)<br />
# Switch-on procedure of components (buttons numbered order)<br />
# Log-in in Windows (user will get an account) and software starting<br />
<br />
'''Stage and objective movement controls''' (XY stage, Z objectives, Switch Objectives on the touch screen - start with the 10X lens. You normally start low, then go higher)<br />
<br />
'''Sample placement''' (sample types, coverslip towards the bottom, adjusting holder, put demo or user sample there, don't clamp it but let it rest (otherwise breaks when objective hits))<br />
<br />
'''Brightfield''' (where does light come from, what do we expect to see)<br />
<br />
'''Focusing''' (sample finding, and then moving around, warning: hitting lens to sample possible)<br />
<br />
'''Epifluorescence''' (see visually light shining, see returning light, discuss different filters, user responsible for knowing their dyes and wavelengths, Searchlight spectra viewer)<br />
<br />
'''Confocal scanning''' (gonna be a big part, mostly on computer now, switch in mood/user to focus on screen not on microscope - instructor sits on the far-right side of the desk to control the mouse, attendees sit right in front of the screen to see everything properly)<br />
<br />
- Launch the software LAS X which controls the microscope, and choose the right startup settings (microscope = DMi8, settings = standard) and wait for it to appear completely. The software is going to ignore any wavelength/imaging modality (BF, EPI) choices you have made using the touch screen of the microscope and do confocal anyway.<br />
<br />
- Overview of software areas:<br />
<br />
# We start in "Acquire" (top) and will be spending most time there<br />
# The big vertical line in the middle divides the view into settings (left) and image display (right)<br />
# Making any adjustments to settings in the image display (right) is not going to change *any* raw data values or *any* microscope settings - just how the image looks on the screen<br />
# Making adjustments on the left will change actual microscope settings, such as wavelengths used, scanning behavior etc.<br />
# At the bottom, there are the buttons that make the microscope actually do something (Live, Capture Image, Start - etc.) - to prevent confusion when clicking "open laser shutter" in the software, or any other buttons.<br />
# We make 1 small excursion to "Configuration" (left of "Acquire") and then "Laser Config" in order to switch on the lasers we know we will need.<br />
<br />
- Laser setup <br />
<br />
# choose the right wavelength<br />
# give it a tiny bit of power (philosophy: start low, go higher if needed - instead of bleaching everything straight away)<br />
# click round button under the laser to open the shutter<br />
# Confirm yellow line appears in the schematic light path below -> laser ready.<br />
<br />
- Detector setup<br />
<br />
# Acknowledge we have a few detectors available, one is switched on.<br />
# Prefer HyD (hybrid detectors), they are better. Typically they are in standard mode with 100 gain (digital gain) and you can use them like that.<br />
# PMT detectors are less sensitive = good for strong signals (usually you have weak signals), to use one you have to give it some gain (couple hundred Volts, until image looks good to you)<br />
# Move the detection window and resize it to capture the desired fluorescence emission wavelengths. Avoid including the laser wavelength at all cost!! This will visualise the laser, which we do not want to see - and it can damage the detector.<br />
<br />
- First image<br />
<br />
# Click "Capture Image"<br />
# Probably, nothing will show up!<br />
# Is there something there, but just weak? -> Set the Image Display (right) to "auto-scale" (button called "M" because it starts on Manual scale) -> have a look if something shows up.<br />
# Is there nothing there anyway, maybe you need more laser -> Increase laser power to 20-30%, capture another image -> have a look if something shows up.<br />
# Is there still nothing, maybe the focus is slightly wrong (we're in confocal mode after all...) -> use the Z-adjustment knob to focus up and down (either on the microscope, on the joystick, or on the USB panel) -> have a look if something shows up, now it really should.<br />
# If there's still nothing, visually check if the sample is still alright, and then use Brightfield or Epifluorescence through the binoculars to find focus again. Then try confocal again, using the software<br />
<br />
- Evaluating the first image<br />
<br />
# It could be too weak (barely jumping out of the noise), so we need more laser until it's visible (improving the scan will help too, but this comes later) at the cost of possibly bleaching<br />
# It could be too strong (huge areas look intense, and same intensity - mouse-over the pixel and see the maximum value 255 show up under the picture = too bright!) you want to avoid saturaiton<br />
<br />
- Optimizing the scan (better picture, takes longer, make it as good as you need and not better)<br />
<br />
- Switching to a higher magnification lens (use navigator to register 1 pic, switch to immersion lens, go back to original location).<br />
<br />
- Z-stack (set start, end, use optimized)<br />
<br />
- Time course (switch modes using dropdown menu, then set (1) interval (none, or amount of time) and (2) duration (unlimited, or amount of time, or amount of pictures)<br />
<br />
- Tilescan (one small one, several of them, focus map)<br />
<br />
<br />
'''Rounding off'''<br />
<br />
- Saving data<br />
<br />
- Saving settings<br />
<br />
- Closing software<br />
<br />
- Shutting down microscope (and cleaning oil objective, then put back at 10X)<br />
<br />
- Leaving room and system tidy for next user</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Leica_SP8_Liachroic_intro&diff=96Leica SP8 Liachroic intro2025-08-06T13:25:24Z<p>Jelle: </p>
<hr />
<div>'''Intro-Intro'''<br />
<br />
# Welcome, outline of the introduction<br />
# The General Instrumentation department conceptually and physically<br />
# The room and the general layout<br />
# The microscope conceptually (functions)<br />
# The microscope physically (components, roughly)<br />
<br />
'''Switch-on'''<br />
<br />
# Quick inspection (everything looks ok visually?)<br />
# Switch-on procedure of components (buttons numbered order)<br />
# Log-in in Windows (user will get an account) and software starting<br />
<br />
Stage and objective movement controls (XY stage, Z objectives)<br />
<br />
Sample placement (sample types, coverslip towards the bottom, adjusting holder, put demo or user sample there, don't clamp it but let it rest (otherwise breaks when objective hits))<br />
<br />
Brightfield mode (where does light come from, what do we expect to see)<br />
<br />
Focusing (sample finding, and then moving around, warning: hitting lens to sample possible)<br />
<br />
Epifluorescence mode (see visually light shining, see returning light, discuss different filters, user responsible for knowing their dyes and wavelengths, Searchlight spectra viewer)<br />
<br />
Confocal scanning mode (gonna be a big part, mostly on computer now, switch in mood/user focus on screen not on microscope)</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Leica_SP8_Liachroic_intro&diff=95Leica SP8 Liachroic intro2025-08-06T13:05:08Z<p>Jelle: made a start</p>
<hr />
<div>'''Introduction'''<br />
<br />
# Welcome, outline of the introduction<br />
# The General Instrumentation department conceptually and physically<br />
# The room and the general layout<br />
# The microscope conceptually (functions)<br />
# The microscope physically (components, roughly)<br />
<br />
Switch-on<br />
<br />
# Quick inspection (everything looks ok visually?)<br />
# Switch-on procedure of components (buttons numbered order)<br />
# Log-in in Windows (user will get an account) and software starting<br />
<br />
Stage and objective movement controls (XY stage, Z objectives)<br />
<br />
Sample placement (sample types, coverslip towards the bottom, adjusting holder, put demo or user sample there, don't clamp it but let it rest (otherwise breaks when objective hits))<br />
<br />
Brightfield mode (where does light come from, what do we expect to see)<br />
<br />
Focusing (sample finding, and then moving around, warning: hitting lens to sample possible)<br />
<br />
Epifluorescence mode (see visually light shining, see returning light, discuss different filters, user responsible for knowing their dyes and wavelengths, Searchlight spectra viewer)<br />
<br />
Confocal scanning mode (gonna be a big part, mostly on computer now, switch in mood/user focus on screen not on microscope)</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Leica_SP8_Liachroic_intro&diff=94Leica SP8 Liachroic intro2025-08-06T12:45:38Z<p>Jelle: created page</p>
<hr />
<div>placeholder</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Leica_SP8_Liachroic&diff=93Leica SP8 Liachroic2025-08-06T12:45:19Z<p>Jelle: </p>
<hr />
<div>Introduction protocol</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Light_microscopy&diff=92Light microscopy2025-08-06T12:45:08Z<p>Jelle: linked to sp8 liachroic</p>
<hr />
<div>This page lists the options for performing light microscopy at the GI department. <br />
<br />
=== List of equipment ===<br />
<br />
==== Light microscopes ====<br />
<br />
* Leica SP8X live-cell confocal<br />
* [[Leica SP8 Liachroic|Leica SP8 liachroic confocal]]<br />
* Leica "Thunder" widefield<br />
* Leica "Timelapse" widefield<br />
* PhaseView Alpha3 light sheet<br />
* Leica DM2500 upright widefield<br />
<br />
* [[Nikon TIRF widefield]]<br />
* [[Nikon sora|Nikon SoRa spinning disc]]<br />
<br />
==== Software packages and other tools ====<br />
<br />
* Spinoza image analysis server<br />
* Huygens<br />
* Microfluidics pump for inverted microscopes<br />
<br />
=== External links ===<br />
[https://bookings.science.ru.nl/category/general-instrumentation-gi/gi-light-microscopy-image-analysis All microscopes (booking system)]</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Leica_SP8_Liachroic&diff=91Leica SP8 Liachroic2025-08-06T12:44:42Z<p>Jelle: created page</p>
<hr />
<div>placeholder</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora_functions&diff=79Nikon sora functions2025-07-28T12:52:21Z<p>Jelle: </p>
<hr />
<div>== What can the Nikon SoRa do? ==<br />
It can do live-cell, dual-color high-speed superresolution confocal imaging, in combination with FRAP<br />
<br />
=== In more detail: ===<br />
<br />
* Live-cell incubation (keeping temperature, humidity and CO2 at desired levels)<br />
<br />
=== Observation by eye, using the binoculars: ===<br />
<br />
* Brightfield (simple white light lamp, light coming from above the objective)<br />
* Widefield epifluorsecence (specific excitation and emission colors, excitation light coming out of the objective)<br />
<br />
=== Observation by camera, using the software: ===<br />
<br />
* Brightfield<br />
* Widefield epifluorescence<br />
* Spinning disc confocal imaging (up to ca. 260 nm resolution)<br />
* Superresolution spinning disc confocal imaging (up to ca. 120 nm resolution)<br />
* FRAP (fluorescence recovery after photobleaching, 488 nm bleaching laser available)</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora&diff=78Nikon sora2025-07-28T12:52:06Z<p>Jelle: </p>
<hr />
<div>[[Nikon sora functions|What can it do?]]<br />
<br />
[[Nikon sora switch on|How to '''switch on''']]<br />
<br />
[[Nikon sora superresolution switch|How to '''switch to superresolution''']]</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora&diff=77Nikon sora2025-07-28T12:51:52Z<p>Jelle: </p>
<hr />
<div>[[Nikon sora functions|What can the Nikon SoRa do?]]<br />
<br />
[[Nikon sora switch on|How to '''switch on''']]<br />
<br />
[[Nikon sora superresolution switch|How to '''switch to superresolution''']]</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora_functions&diff=76Nikon sora functions2025-07-28T12:50:33Z<p>Jelle: </p>
<hr />
<div>== What can the Nikon SoRa do? ==<br />
It can do live-cell, dual-color high-speed superresolution confocal imaging, in combination with FRAP<br />
<br />
=== In more detail, it can do: ===<br />
<br />
* Live-cell incubation (keeping temperature, humidity and CO2 at desired levels)<br />
<br />
=== Observation by eye, using the binoculars: ===<br />
<br />
* Brightfield (simple white light lamp, light coming from above the objective)<br />
* Widefield epifluorsecence (specific excitation and emission colors, excitation light coming out of the objective)<br />
<br />
=== Observation by camera, using the software: ===<br />
<br />
* Brightfield<br />
* Widefield epifluorescence<br />
* Spinning disc confocal imaging (up to ca. 260 nm resolution)<br />
* Superresolution spinning disc confocal imaging (up to ca. 120 nm resolution)<br />
* FRAP (fluorescence recovery after photobleaching, 488 nm bleaching laser available)</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora_functions&diff=75Nikon sora functions2025-07-28T12:49:30Z<p>Jelle: </p>
<hr />
<div>What can the Nikon SoRa do?<br />
<br />
Briefly: It can do live-cell, dual-color high-speed superresolution confocal imaging, in combination with FRAP (optional).<br />
<br />
In more detail, it can do:<br />
<br />
- Live-cell incubation (keeping temperature, humidity and CO2 at desired levels)<br />
<br />
Observation by eye, using the binoculars:<br />
<br />
- Brightfield (simple white light lamp, light coming from above the objective)<br />
<br />
- Widefield epifluorsecence (specific excitation and emission colors, excitation light coming out of the objective)<br />
<br />
Observation by camera, using the software:<br />
<br />
- Brightfield<br />
- Widefield epifluorescence<br />
- Spinning disc confocal imaging (up to ca. 260 nm resolution)<br />
- Superresolution spinning disc confocal imaging (up to ca. 120 nm resolution)<br />
- FRAP (fluorescence recovery after photobleaching, 488 nm bleaching laser available)</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora_functions&diff=74Nikon sora functions2025-07-28T12:49:17Z<p>Jelle: setting up page</p>
<hr />
<div>What can the Nikon SoRa do?<br />
<br />
Briefly: It can do live-cell, dual-color high-speed superresolution confocal imaging, in combination with FRAP (optional).<br />
<br />
In more detail, it can do:<br />
<br />
- Live-cell incubation (keeping temperature, humidity and CO2 at desired levels)<br />
<br />
Observation by eye, using the binoculars:<br />
<br />
- Brightfield (simple white light lamp, light coming from above the objective)<br />
- Widefield epifluorsecence (specific excitation and emission colors, excitation light coming out of the objective)<br />
<br />
Observation by camera, using the software:<br />
<br />
- Brightfield<br />
- Widefield epifluorescence<br />
- Spinning disc confocal imaging (up to ca. 260 nm resolution)<br />
- Superresolution spinning disc confocal imaging (up to ca. 120 nm resolution)<br />
- FRAP (fluorescence recovery after photobleaching, 488 nm bleaching laser available)</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora_switch_on&diff=73Nikon sora switch on2025-07-28T07:52:52Z<p>Jelle: /* Required elements: */</p>
<hr />
<div>[[File:Sora_schematic_switch_on.png|1000x1000px]]<br />
<br />
=== Required elements: ===<br />
1) '''Controller''': powers the microscope stand<br />
<br />
2) '''Temp. incubation''' - even if you don't need temp incubation; switch off the heater using its button on this box<br />
<br />
5) '''Laser box''' - lasers are required to image<br />
<br />
7) '''Cam 1''' - at least 1 camera is required to image<br />
<br />
9) '''SoRa unit''' - this is required for confocal imaging<br />
<br />
10) '''PC''' - required for using the camera(s), multidimensional acquisitions, any confocal experiments<br />
<br />
=== Optional elements: ===<br />
3) '''Humidity + CO2''' - only when live-cell imaging samples that require this controlled - make sure to add water to the incubator<br />
<br />
4) '''FRAP scanner''' - only when intending to do [[wikipedia:Fluorescence_recovery_after_photobleaching|FRAP]] (fluorescence recovery after photobleaching) experiments<br />
<br />
6) '''FRAP interlock -''' only when intending to do FRAP experiments<br />
<br />
8) '''Cam 2''' - only when intending to do simultaneous 2-color imaging experiments</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora_switch_on&diff=72Nikon sora switch on2025-07-28T07:51:07Z<p>Jelle: </p>
<hr />
<div>[[File:Sora_schematic_switch_on.png|1000x1000px]]<br />
<br />
=== Required elements: ===<br />
1) '''Controller''': powers the microscope stand<br />
<br />
2) '''Temp. incubation''' - even if you don't need temp incubation; switch off the heater using its button on this box<br />
<br />
5) '''Laser box''' - you need lasers to image<br />
<br />
7) '''Cam 1''' - you need at least 1 camera to image<br />
<br />
9) '''SoRa unit''' - this is required for confocal imaging<br />
<br />
=== Optional elements: ===<br />
3) '''Humidity + CO2''' - only when live-cell imaging samples that require this controlled - make sure to add water to the incubator<br />
<br />
4) '''FRAP scanner''' - only when intending to do [[wikipedia:Fluorescence_recovery_after_photobleaching|FRAP]] (fluorescence recovery after photobleaching) experiments<br />
<br />
6) '''FRAP interlock -''' only when intending to do FRAP experiments<br />
<br />
8) '''Cam 2''' - only when intending to do simultaneous 2-color imaging experiments</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora_switch_on&diff=71Nikon sora switch on2025-07-28T07:50:47Z<p>Jelle: added required/optional elements</p>
<hr />
<div>[[File:Sora_schematic_switch_on.png|1000x1000px]]<br />
<br />
=== Required elements: ===<br />
'''1) Controller''': powers the microscope stand<br />
<br />
'''2) Temp. incubation''' - even if you don't need temp incubation; switch off the heater using its button on this box<br />
<br />
'''5) Laser box''' - you need lasers to image<br />
<br />
'''7) Cam 1''' - you need at least 1 camera to image<br />
<br />
'''9) SoRa unit''' - this is required for confocal imaging<br />
<br />
=== Optional elements: ===<br />
'''3) Humidity + CO2''' - only when live-cell imaging samples that require this controlled - make sure to add water to the incubator<br />
<br />
'''4) FRAP scanner''' - only when intending to do [[wikipedia:Fluorescence_recovery_after_photobleaching|FRAP]] (fluorescence recovery after photobleaching) experiments<br />
<br />
'''6) FRAP interlock -''' only when intending to do FRAP experiments<br />
<br />
'''8) Cam 2''' - only when intending to do simultaneous 2-color imaging experiments</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora&diff=70Nikon sora2025-07-08T13:29:45Z<p>Jelle: </p>
<hr />
<div>[[Nikon sora switch on|How to '''switch on''']]<br />
<br />
[[Nikon sora superresolution switch|How to '''switch to superresolution''']]</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora&diff=69Nikon sora2025-07-08T13:29:06Z<p>Jelle: </p>
<hr />
<div>[[How to switch on]]<br />
<br />
[[Nikon sora superresolution switch|How to switch to superresolution]]</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora_switch_on&diff=68Nikon sora switch on2025-07-08T13:28:50Z<p>Jelle: </p>
<hr />
<div>[[File:Sora_schematic_switch_on.png|1000x1000px]]</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora_switch_on&diff=67Nikon sora switch on2025-07-08T13:28:05Z<p>Jelle: Created page with "thumb"</p>
<hr />
<div>[[File:Sora schematic switch on.png|thumb]]</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=File:Sora_schematic_switch_on.png&diff=66File:Sora schematic switch on.png2025-07-08T13:28:01Z<p>Jelle: </p>
<hr />
<div>-</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora&diff=65Nikon sora2025-07-08T13:26:42Z<p>Jelle: </p>
<hr />
<div>How to switch on<br />
<br />
[[Nikon sora superresolution switch|How to switch to superresolution]]</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Light_microscopy&diff=57Light microscopy2025-07-04T15:03:29Z<p>Jelle: </p>
<hr />
<div>[https://bookings.science.ru.nl/category/general-instrumentation-gi/gi-light-microscopy-image-analysis All microscopes (booking system)]<br />
<br />
<br />
<br />
[[Nikon TIRF widefield]]<br />
<br />
[[Nikon sora|Nikon SoRa spinning disc]]</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Light_microscopy&diff=56Light microscopy2025-07-04T15:02:19Z<p>Jelle: </p>
<hr />
<div>[https://bookings.science.ru.nl/category/general-instrumentation-gi/gi-light-microscopy-image-analysishttps://bookings.science.ru.nl/category/general-instrumentation-gi/gi-light-microscopy-image-analysis All microscopes (booking system)]<br />
<br />
<br />
[[Nikon TIRF widefield]]<br />
<br />
[[Nikon sora|Nikon SoRa spinning disc]]</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora&diff=55Nikon sora2025-07-04T13:57:19Z<p>Jelle: </p>
<hr />
<div>[[Nikon sora superresolution switch|How to switch to superresolution]]</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Light_microscopy&diff=54Light microscopy2025-07-04T13:56:57Z<p>Jelle: </p>
<hr />
<div>[[Nikon TIRF widefield]]<br />
<br />
[[Nikon sora|Nikon SoRa spinning disc]]</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora_superresolution_switch&diff=53Nikon sora superresolution switch2025-07-04T13:56:19Z<p>Jelle: </p>
<hr />
<div>How to switch from normal to superresolution:<br />
# Use the '''100x / 1.49 oil''' objective (is the highest numerical aperture lens available)<br />
# Switch to the '''SoRa spinning disc''' under "pinhole" <br />
# Switch to the '''2.8x SoRa magnifier'''<br />
# Set the camera to "'''binning 1x1'''" (no binning)<br />
# '''Obtain a Z-stack''': Make the stack slightly "too big" - having some extra blurry layers above/below the structure helps with deconvolution afterwards.<br />
# Perform "'''3D Deconvolution'''" (software command) with the standard suggestged settings (mostly change "noisyness" of the picture when needed)<br />
# '''Evaluate''' result and adjust parameters where necessary (e.g. adjust Z-step size, adjust image "noisyness" setting upon deconvolution)<br />
How to switch back to normal imaging again:<br />
<br />
# Switch back to '''50 um''' pinhole (not SoRa)<br />
# Switch back to the '''1x''' '''SoRa magnifier''' (not 2.8x or 4.0x)<br />
# Taking z-stacks/using no camera binning is not essential anymore here</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora_superresolution_switch&diff=52Nikon sora superresolution switch2025-07-04T13:53:46Z<p>Jelle: </p>
<hr />
<div>How to switch from normal mode<br />
# Use the '''100x / 1.49 oil''' objective (is the highest numerical aperture lens available)<br />
# Switch to the '''SoRa spinning disc''' under "pinhole" <br />
# Switch to the '''2.8x SoRa magnifier'''<br />
# Set the camera to "'''binning 1x1'''" (no binning)<br />
# '''Obtain a Z-stack''': Make the stack slightly "too big" - having some extra blurry layers above/below the structure helps with deconvolution afterwards.<br />
# Perform "'''3D Deconvolution'''" (software command) with the standard suggestged settings (mostly change "noisyness" of the picture when needed)<br />
# '''Evaluate''' result and adjust parameters where necessary (e.g. adjust Z-step size, adjust image "noisyness" setting upon deconvolution)</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora_superresolution_switch&diff=51Nikon sora superresolution switch2025-07-04T13:52:22Z<p>Jelle: </p>
<hr />
<div># Use the '''100x / 1.49 oil''' objective<br />
# Enable the '''2.8x SoRa magnifier'''<br />
# Set camera to "'''binning 1x1'''" (no binning)<br />
# '''Obtain a Z-stack''': Make the stack slightly "too big" - having some extra blurry layers above/below the structure helps with deconvolution afterwards.<br />
# Perform "'''3D Deconvolution'''" (software command) with the standard suggestged settings (mostly change "noisyness" of the picture when needed)<br />
# '''Evaluate''' result and adjust parameters where necessary (e.g. adjust Z-step size, adjust image "noisyness" setting upon deconvolution)</div>Jellehttps://wiki.gi.science.ru.nl/index.php?title=Nikon_sora_superresolution_switch&diff=50Nikon sora superresolution switch2025-07-04T13:49:48Z<p>Jelle: first add</p>
<hr />
<div># 1) Use the 100x / 1.49 oil lens<br />
# 2) Enable the 2.8x SoRa magnifier<br />
# 3) Set camera to "binning 1x1" (no binning)<br />
# 4) Obtain a Z-stack: Include lots of blur above / below the interesting focus layers<br />
# 5) Perform "3D Deconvolution" (software option) with the standard settings.<br />
# 6) Evaluate result and adjust parameters where necessary (e.g. adjust Z-step size, adjust image "noisyness" setting upon deconvolution)</div>Jelle