Fluorescence microscope & cooled CCD camera

Responsible: Anders Liljeborg

Short user guide

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Overview of the fluorescence microscope with cooled CCD camera

NEW! Scale-bars for the different objectives and magnifications

This is the fluorescence microscope equipped with a cooled CCD camera.
From left to right are shown:

The microscope is equipped with four objectives:
(wd = working distance, NA = Numerical Aperture, NCG = No Cover Glass)

Warning!

The Mercury lamp must burn for at least one hour when turned on. Typically it should not be turned off for a lunch-break, it should be left running.
This is because certain vapors in the lamp can form stable residues on the inside of the lamp-glass, thus reducing the light intensity and the life span of the lamp.

The 100x/1.4 NA oil-immersion objective should always be used with a drop of immersion oil on the specimen. Wipe the objective with lens-cleaning paper after use. immersoil-sm.jpg

The CCD camera is monochrome and has an optical resolution of 1600 × 1200 7.4 µm square pixels. Image area is 11.8 × 8.9 mm.

The power supply for the mercury vapor lamp.
  • At the top is the hour counter for the burn-time of the lamp.
  • Power LED and mercury lamp ready indicator
  • Ignition button
  • Mains power switch
When turning on the lamp:
  • first turn on the power switch
  • then press and hold down the ignition button until the orange "Lamp Ready" is lit and the run time counter has advanced one tenth-hour step.

Warning!

The Mercury lamp must burn for at least one hour when turned on. Typically it should not be turned off for a lunch-break, it should be left running.
This is because certain vapors in the lamp can form stable residues on the inside of the lamp-glass, thus reducing the light intensity and the life span of the lamp.

When turning off the mercury lamp; turn off the power switch.
Note: there is no way to vary the intensity of the mercury lamp electrically, this must be done with grey-filters.

The power supply for the CCD camera. A simple On/Off switch, just turn it on at the beginning of the session, turn it off when you are finished.



This is the center of the microscope, the epi-illumination fluorescence unit.

The slider with four positions is controlling which filter block is in the light path, thus selecting which type of fluorescence to detect.

Assisting here is a bandpass filter located in the black holder directly above the slider. The filter is labelled "XF 3080 510AF23 466 0130". This means 510 nm center wavelength and 23 nm full-width-half-maximum. It is a narrow band filter to help reduce sample auto-fluorescence. Can also be helpful for discrimination from red emitting fluorophores such as mRFP. Should be used together with position #1, possible fluorophores eGFP, Fluorescein (FITC), Alexa Fluor 488, Cy2.

At the top right corner there is a shutter to block the excitation light from the specimen when not looking at it. This is a very useful item since the specimen can easily be bleached by the rather powerful mercury lamp.



The grey filters (neutral density filters) to vary the intensity of the illumination/excitation light.

There are three grey filters used to vary the intensity of the illumination/excitation light. These are labelled:

The filters are used by pushing them into the light path in any combination.


The objectives in their revolver, the specimen stage and the focusing knobs.

Here is the stage where a Si-chip layered with gold is studied. Note the little bluish spot where the excitation light illuminates the chip.
At the lower left is the focusing knob:

At the lower right can be seen the knobs for moving the stage laterally.


The stage and focus knobs seen from the left side.

Here knobs for moving the stage are more clearly seen.
Behind them is a dual knob. The outer part is a fine focus knob so fine focus can be adjusted with either hand.
The inner ring is a locking ring which stops the coarse focus from moving the stage above a certain level. This can be very useful to protect oneself from pushing the specimen into the objective:

Now the stage cannot be moved upwards more than for focusing, it stops before the specimen touches the objective. To release the lock just loosen the locking ring.


Here is the housing of the mercury lamp.
It can become quite hot, so do not touch it. There are several adjustment screws for aligning the lamp when it has been replaced. These screws should not be touched without knowledge.
To the left is the control for the field stop. It can be used to limit the circular field that is illuminated on the specimen. The black knob adjusts the size (diameter) of the field, the two blank metal knobs adjust the centering of the field inside the field-of-view.


At the top of the microscope the CCD camera is attached.

The CCD camera is a SPOT RT Monochrome manufactured by Diagnostics Instrument. It has a CCD made by Kodak, KAI - 2000, with protective cover glass removed. Optical resolution is 1600 × 1200 7.4 µm square pixels. Image area is 11.8 × 8.9 mm.
Bit depth: 8 or 12 bits, binning: 2×2, 3×3, 4×4.
CCD cooling: 37° differential from room temperature (-12°C at 25°C room temperature). Stability: ± 1°C per 8 hour period.
Dark current: 0.15 electrons per pixel per second (e/p/s) at -12°C (typical).
Full scale charge: 35,000 e¯.
Signal to Noise Ratio: 46 dB, limited by photon quantum noise (Poisson statistics).
Dynamic range: 60 dB.
Anti-blooming factor: 300 times (nominal), 100 times (minimum).
Quantum efficiency see diagram:

There is also a camera window affecting the spectral response of the camera. It is made of UBK7 glass with 320 - 900 nm anti reflective coating. See transmission diagram:


Finally we can see the operator interacting with the microscope.

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Thinking a bit of how the specimen is being imaged.

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An in-depth study.


Anders Liljeborg Nano-Fab-Lab, KTH. Featured operator: Jonas Rundqvist