Just Getting started UV spectrometry microscopy

[dabateman]

I have been playing around with connecting my spectrometer in line with a microscopy and had my first success.

The spectrometer is connected to the eye piece using an adapter I made which runs to two channels. The red is a spectrometer with grating #1 and can see from 175nm to 869nm. The blue curve is a spectrometer that can see from 500nm to 1150nm.

 

For this I have a piece of paper on my microscope as a control with green highlighter on it. Here is image taken with 10x and lodestar X2c cammera;

 

The bright purple is the reflected 365nm filtered convoy off just the paper. the bright streak is green highlighter marked on the paper.

 

This is the resulting spectrum grab screen shot from the raw data:

 

 

This actually follows in the focal plane. I loose signal when out of focus and its back with in focus. So I am paralax. I should now be able to see the spectal output for specific cells.

 

Great first steps.

[Stefano]

It seems like the second spectrometer has a 500 nm longpass filter in it. Probably it isn’t a good idea to remove it, it surely isn’t designed to measure below 500 nm.

[dabateman]

The blue curve one has a 475nm long pass filter in the 25um slit. I had it set up for fluorescence, but my other module is 10x more sensitive.

 

[JMC]

That's really funky. Do you know how much of the field of view the spectrometer is capturing data from David?

[SteveE]

The long pass filter is to suppress second order spectra in the IR region, at least part of it. A spectral line at 500um will produce a false second order spectra at 1000um. The low pass filter will suppress second order spectra down to about 950um or so (assuming perfect cutoff at 475um). Ocean Optics has second order filters available that are on the sensor, but they don't seem to have a sensor filter that goes down into the infrared (at least I've never seen one), so they will typically use low pass filters on the input.

[dabateman]

That's really funky. Do you know how much of the field of view the spectrometer is capturing data from David?

 

I don't know. I will have to test that with a pin point of highlighter and move it around my field in xy space.

 

I was just excited it works in z space. I didn't want the full cube (voxel) of available light being detected. My modification is an eyepiece to m42 adapter, the double female m42 adapter then spacer and a cone to SMA connection inside a M42 to 1.25" adapter. Just at the mouth of the eyepiece adapter I am using my fused silica 25mm Bicx lens in a c-mount.

I should take a photo.

 

This hopefully will let me tease out many different fluorophores based on their different tails. Back to my 8 channel flow cytometry days. Should be fun.

Still have to test out some cells now.

[dabateman]

Well here you go, a femtosecond capable system that has been developed.

https://petapixel.com/2021/07/14/scientists-develop-camera-system-that-captures-5d-images/

 

Funny if I worked out the region of optical alignment that my microscope was providing, I may still be able to publish this as lower cost solution.

 

The microscope optics are my beam splitter. My spectrometer is good but slow compared to this.

 

For this publication, I would have liked to see some live blood cell motion.