Laser Confocal Microscope Specifications

1. Optical and scanning system

• Infinity corrected optics with 45 or 60 mm parfocal distance

• Point scanning by galvo mirrors as standard, spinning disk for fast live cell work

• Scan head integrated with the stand or separate, the latter being easier to upgrade

• Light path supporting brightfield, fluorescence and confocal modes

2. Resolution and imaging performance

• Lateral resolution around 200 nm with a 60X 1.4 oil objective at 488 nm excitation

• Axial resolution around 500 to 700 nm

• Scan resolution up to 4096 by 4096 pixels

• A pinhole at 1 Airy unit balances resolution and light throughput, smaller means thinner slices and weaker signal

• Note: resolution follows objective numerical aperture and wavelength, and quoted figures come from the highest aperture oil objectives

3. Lasers and detection channels

• Solid state lasers, commonly 405, 488, 561 and 640 nm, at 10 to 50 mW output

• Laser power adjustable in software, with acousto-optic modulation or neutral density filters

• Three to four detection channels with photomultiplier or GaAsP detectors

• Spectral detection with up to 32 channels for unmixing overlapping fluorophores

• Simultaneous multichannel imaging requires attention to crosstalk and scan delay

4. Scan modes and speed

• Standard scanning at 512 by 512 giving 1 to 2 frames per second

• Bidirectional and resonant scanning raising this to 4 to 30 frames per second depending on model

• Zoom from 1 to 32 times, enlarging a region of interest without losing resolution

• Fast dynamics such as calcium imaging or blood flow need a resonant head or spinning disk

• Speed trades against signal-to-noise, and fast scanning usually needs more laser power or binning

5. Pinhole and optical sectioning

• Continuously adjustable pinhole from 0 to 1000 micrometres

• Pinhole set in Airy units, one unit equalling one optical section thickness

• Z step of 10 nm or less with a piezo stage, enabling Z stacks and 3D reconstruction

• Section thickness around 0.8 micrometres with a high aperture oil objective and above 5 at low power

• Z drift compensation or autofocus is essential for long live cell runs

6. Stage and live cell incubation

• Motorised X Y stage with at least 100 by 75 mm travel and 1 micrometre repeatability

• Accepting 35 mm dishes, multiwell plates, chamber slides and standard slides

• Incubation holding 37 degrees Celsius within 0.5 and CO2 at 5 percent within 0.2

• Multipoint time lapse across multiwell plates

• Objective working distance must still be respected once the chamber is fitted

7. Software and image analysis

• Multichannel acquisition, channel merging and false colour mapping

• 3D reconstruction by maximum intensity projection, orthogonal views and surface rendering

• Colocalisation analysis with Pearson and Manders coefficients

• Fluorescence intensity quantification, area and morphology measurement, particle counting

• Deconvolution and large area stitching

• Export to TIFF, OME-TIFF and AVI

8. Power and environment

• Power supply AC 220 V, 50 Hz, with a stabiliser or UPS recommended

• Operating temperature 20 to 25 degrees Celsius, drift within 1 degree per hour

• Relative humidity up to 70 percent, avoiding condensation

• Anti-vibration table, air or marble, away from lifts, compressors and corridors

• Darkened or controlled lighting so ambient light does not enter the detection path

9. Where procurement goes wrong

• Only confocal microscope is specified without laser wavelengths or channel count, and two channels arrive, making double labelling impossible

• No incubation chamber and no autofocus, so cells drift out of focus during time lapse and the data are lost

• The stage does not match the vessels in use, so a 96 well plate cannot be imaged well by well