Leica DM6B Manuel utilisateur

Guide for Fluorescence Imaging

1. Initial Set-up

~ Log in to NUCore and start a reservation.

~ Turn on Microscope Controller (A).

~ Turn on LED main power (B) and press control knob (C) to see a flashing

sign.

~ Wait the microscope initialization to complete and launch LASX

software. (Computer has no password).

~ Put slide on scope and set up image acquisition.

2. Mount sample on microscope

~ Place slide on stage ‘coverslip up’.

~ For transmitted light imaging:

Select BF button (D) and press TL-Shutter (F) on LED screen. Focus

on sample.

~ For fluorescent light imaging:

Select FLUO (E), then appropriate filter cube from buttons on LED

screen (DAP –Blue, L5 –Green, RHO –Red, Y5 –Far Red), open IL-

Shutter (F).Focus on sample.

~ Find cell/region of interest. Close shutter.

NOTE: To switch light path from oculars to camera gently pull out slider

bar located in microscope head near oculars on the left side.

3. Troubleshooting

~ No fluorescent emission by eye?

Check the LED is ON. Control knob should show

Check that light path is going to the oculars. The slider bar microscope head should be inside.

~ No image displayed on a screen in LIVE mode?

Check that light path is going to the camera. The slider bar microscope head should be taken out.

~ Still having problems?

Contact BIF staff for assistance.

Leica DM6B Widefield

Quick Start Guide

4. Setting up Fluorescent light imaging

~ In software, navigate to

“Acquisition” tab (G). Select

required Image Format and Live

Format (binning 1x1, 2x2 etc)

and “Black & White mode” for

the camera (H).

~ Then, on “Acquire”tab (I), set

up light path for imaging of

fluorophores of interest (J).

Press “+” or “-” button to achieve

necessary number of channels

(one for each fluorophore to be

imaged). The selected channel is

colored red. For each channel,

select from preset settings (dropdown list) or manually set by selecting “FLUO”, then selecting the

appropriate filter set (hover over filter for details) and set LUT by colored bar at top of channel.

~ Set image acquisition parameters for each fluorophore, as appropriate. To do that, select the one to be

adjusted. Then click the “Live” button to check settings.

~ View image as intensity values by selecting LUT (Look Up Table) tool with the QLUT button.

~ Adjust Exposure and Gain (K) so that there are mostly orange and red pixels and no blue (saturated)

pixels. Click “Stop”. Gain should be below 4 for highest quality image. Switch to another channel and

repeat adjustments.

~ When all settings are set, click "Start" to start the acquisition.

5. Acquiring a Z-series

~ In the Acquisition tab (G), be sure X, Y & Z are selected under Acquisition Mode.

~ Click “Live”. Move to the top of your sample with the focusing knob and set the position by clicking the

“Begin” arrowhead (L).

~ Move to the bottom of your sample with the focusing knob and set the position by clicking on the “End”

arrowhead (L). Click “Stop”.

~ Set the desired number/size of z steps.

~ Ensure imaging settings are correct, then hit “Start” to collect z-stack.

6. Save images

~ In the “Open projects” tab, click “Save All”. Select the appropriate

directory and change the Experiment file name, if desired. Click OK to save.

7. Shut Down

~ Remove sample. Clean oil off objectives with lens paper. Return objective turret to 10x.

~ Transfer files to flash drive or network storage. Close software.

~ If there is another user coming within 1 h, stop here, leaving microscope ON.

~ If not, proceed with following steps for complete shutdown:

~ Turn OFF the microscope controller box (A) and LED box (B). Cover microscope.

~ End reservation in NUCore. If help is needed, please see BIF staff

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