Beamsplitter Plugs and Microscope Cubes for Photostimulation
Before proceeding with calibration you should ensure the correct microscope cubes and beamsplitter plugs are installed in the Mosaic to ensure delivery of imaging and photostimulation wavelengths to the specimen.
Beamsplitter Plugs
Beamsplitter plugs are exchangeable inserts for the Mosaic (and MicroPoint) photostimulation products that contain a mirror or dichroic. The purpose of these as the name infers, is to control the wavelengths that pass through directly (transmission), and a second source from the side (reflection) making it possible for different light sources to be combined with the Mosaic for photostimulation applications. The Mosaic can have up to 2 Beamsplitter blocks present:
One in an input block for photostimulation illumination sources
The other for EPI illumination.
The beamsplitter plugs are interlocked and must be in place for laser illumination. Beamsplitters are normally specified at the time of ordering so that suitable beamsplitters are installed to suit the expected applications. The beamsplitters may be as follows:
Blank: All of the light passes directly through the beamsplitter for maximum transmission. Typically used when you want maximum transmission.
Bandpass: Dichroic filter that may be a short pass or long pass. For example, a 405nm longpass would reflect wavelengths at 405nm or below and transmit wavelengths above 405.
Notch: These wavelength filters block a narrow band of the light spectrum, while allowing other wavelengths on either side to pass through.
% mirror: Mirror Beamsplitters are typically used to provide a balance between 2 light sources across all wavelengths. For example, you could combine Photostimulation wavelengths from Mosaic at the EPI illumination block using different beamsplitters to vary the relative amount from the Mosaic (Transmission value) and the EPI illumination for visualisation from the eyepieces (Reflect value).
50/50 would provide a 50/50 split between the output of the Mosaic which passes directly to the microscope port, and the eyepiece illumination from the side
70/30 would allow 70% transmission from the Mosaic, and a reduced 30% (reflection) for the eyepiece illumination
30/70 would allow only 30% transmission from the Mosaic, and increase this to 70% (reflection) for the eyepieces
0/100 (100% reflect) mirror would allow 100% epi illumination and no output from the Mosaic when installed in the EPI illumination block.
The normal recommendation is to use a blank for the photostimulation input block so that all the light is passed through the Mosaic. For more complex configurations using multiple light sources such as an HLE and a Mosaic Diode laser, other Beamsplitter options may be required to combine both wavelengths in a single protocol.
For the EPI input block (if present), a 70/30 beamsplitter is often fitted as 70% of the photostimulation is transmitted, and some 30% of the EPI light from an LED source is available for visualisation through the eyepieces. For further assistance please contact your local support representative.
Microscope Cubes
The Microscope cube is a critical part of the lightpath for photostimulation. It is essential that a suitable microscope cube is selected that will permit photostimulation and imaging. As with beamsplitter plugs, microscope cube(s) for photostimulation will have been specified when the Mosaic was ordered. The function of the microscope cube in a photostimulation experiment is to reflect the photostimulation wavelength, and transmit the imaging wavelength(s).
While it is possible to setup the microscope cube in a photostimulation channel, and use a different microscope cube in an imaging channel this would mean that the cube would switch in the protocol for imaging, then photostimulation and again for imaging each time. Since many photostimulation applications happen in very short time scales this delay would not be desirable. Therefore, it is normally recommended to use the same microscope cube for imaging and photostimulation within the protocol. Some common examples and their application are outlined below (note that part numbers and descriptions are within the Mosaic Manual).
405 Longpass: for photostimulation at 405nm and imaging at wavelengths >405nm. Note that the Mosaic is not recommended for photostimulation applications below 400nm as the response of the Mosaic starts to drop sharply as may the objective lens.
460 Longpass: for photostimulation below 460nm and imaging above 460nm.This would permit photostimulation of wavelengths such as 405 or 445nm and imaging of GFP and other flurophores above 460nm.
Mirror Cubes – as with the beamsplitters, these allow flexibility for use with a broad range of wavelengths. Mirror cubes are available with different % splits between photostimulation and imaging wavelengths. These are not optimal with a carefully selected bandpass filter, as you are blocking some degree of the light e.g. for a 50/50 mirror cube, 50% of the photostimulation and 50% of the imaging wavelengths will pass through. The mirror cubes are flexible, and still provide sufficient throughput for many applications. A 70/30 mirror cube would be a recommendation that would give more photostimulation power.
Other cubes may be specified for custom or specific applications that require photoactivation or conversion at higher wavelengths. Refer to the Mosaic User Guide or specification sheets for a full description of Microscope cube product numbers and their descriptions to confirm their spectral response.
Suitability of Objectives
A further consideration before calibration is the objective. The objective plays a key part in the lightpath and important to photostimulation being effective. As a general guide most objectives including air and oil immersion, are suitable for photostimulation. The important parameters to check are as follows:
Spectral Response (transmission): objectives can have poor transmission towards the UV region and this can drop off quite sharply. Check the transmission for your objective especially when performing photostimulation at 405nm, but some objectives can have poor transmission below 480nm. Ideally you would want an objective with a & transmission higher than 70% at the wavelength to be used. This is important as the Mosaic, camera and other optics within the system may also have a drop in response around this region and the resulting final power density at the specimen and what is observed may be low. Note that objectives with different magnification within the same product range can have varying spectral response.
Objective NA and Magnification: Higher NA objectives will provide higher resolution of the photostimulation illumination at the specimen than lower NAs for the same magnification. Higher magnification, high NA objectives can allow finer structures within cells such as vesicles be targeted with greater precision. Higher magnifications may also allow provide greater power density- for example a 40x objective can have ~4x more power density than if using a 20x objective. This will depend on the characteristics of the objectives. The downside of a higher magnification is reduced field of view.