Notes on near IR microscopy with a tungsten lamp and visible light blocking filters

 by David Walker, UK.

As for many hobbyists, the advent of cheap LEDs used with suitable consumer digicams has prompted my interest in digital photomicroscopy just outside the visible light spectrum. Both at shorter wavelengths (near UV) for enhanced resolution and longer wavelengths (near IR) for superior light transmittance with near opaque subjects like insect exoskeletons.

To date, I've used near infra red (NIR) on my LOMO microscope using its external tungsten lamp and an LED soldered onto a blown bulb stub with a suitable low voltage power supply (not mains!). Near IR, near UV and violet LEDs on blown bulbs allow quick interchange for multi-wavelength studies.

More recently I've been trying a Zeiss Photomicroscope for near IR and UV studies. I've also tried LEDs on a blown bulb stub for the 12V 60W lamphouse, but unlike the external LOMO lamp, readily interchanging between LED and tungsten lighting was inconvenient. So as an alternative I tried filters to block visible light from the tungsten lamp but pass the NIR component. A reader kindly remarked that this technique can work well from their own experiences but hadn't tried it to date. Experiences using two types of blocking filters are summarised below as they may be of interest to others wishing to try NIR with tungsten lamps.

Hoya R72 filter: This filter or equivalent is commonly used in conventional near IR photography. With a bright microscope lamp it transmits a visible deep crimson red and near IR (50% transmittance at 720 nm). The full transmittance curve can be viewed on the Hoya website. Purchasing tip: I already had a Hoya R72 52 mm lens grade filter for normal NIR landscape photography, but these are expensive. A cheaper optical grade and smaller area can be used for microscopy as the filter sits on the field iris below the microscope objectives.

Total visible light blocking filters: I'd used a 940 nm NIR LED to date so was interested in a filter that gave deeper NIR than the R72. I bought an 850 nm 'long pass filter' from www.knightoptical.co.uk (a 5 cm² acrylic filter was less than £10). From specs supplied by Knight Optical, this filter had a sharp cut off, passing no light at all < 800 nm and 50% at 850 nm (i.e. a similar transmittance to that reported for a Hoya IR85 filter). A tungsten lamp at a typical filament temp has excellent emission below 700 nm into the near IR wavelengths (see ref. 1).

The author used both an OpticStar 1.3 Mpixel 'C' mount monochrome camera (sold for astronomy) and a Nikon D50 DSLR. There's many resources on the web discussing which unmodified digicam models are most suitable for NIR. From browsing web camera forums, my relatively old D50 seems to be regarded as 'weakly filtered' for NIR compared with newer models like the D300. The live video feed from the OpticStar and similar cameras could be used instead of stills for demonstration purposes.

Compound microscope (Zeiss photomicroscope III).

1.3 Mpixel 'C' mount CMOS monochrome USB2.0 camera. Opticstar PL-130M.
Diving beetle, Zeiss 1x NA0.04 planachro objective. Prepared by the author on the late Eric Marson's course. 

Nikon D50 DSLR. Out of camera resized images. ISO 200. Zeiss 12V / 60W lamp at 12 Volts.
Diving beetle, Zeiss 1x NA0.04 planachro objective. Prepared by the author on the late Eric Marson's course.

No filter on lamp i.e visible brightfield in monochrome. Little to no detail seen in the exoskeleton. 1/1250 sec. Increasing the lamp intensity just blows out the highlights, without revealing detail in the insect.

Hoya R72 filter on field iris. Converted to monochrome, the master is a deep red. Same lamp setting, ND1.0 light reduction, 1/13th sec. Detail now seen in exoskeleton apart from the thickest areas. The filter gives a deep crimson image which can be focussed visually.

850 nm long pass filter on field iris. 4 sec. Although 6 - 7 stops greater exposure was required cf. the R72, sensitivity of the D50 was still good and detail is seen even in the thickest areas. No visual image is seen but possible to calibrate each objective's new focus position with a camera that gives live image. (For example, with the Zeiss 2.5x objective on the photomicroscope, 3.5 revolutions up on the fine focus position for white light was the near IR focus.)
The limited tonal range of a NIR image at this and longer wavelengths seems fairly typical from the author's experiences with the D50 or other digicams tried, so the images are more informative than artistically pleasing. Depending on subject and degree of internal detail that can be revealed, the more contrasty R72 filter may be preferred.

Not surprisingly, the D50 is much more sensitive to wavelengths of 720 or 850 nm compared with the 940 nm LED used previously, where very high ISO and long exposures were needed. The author briefly assessed the recent Nikon D300 model using the same filters and found that depending on wavelength it was 3 - 7 stops less sensitive for transmitted NIR than the D50. Discussions in online camera forums seems to suggest that modern DSLRs are often using more extensive NIR blocking.

Stereo microscope
The large insect slides are perhaps better suited for wider field imaging on a stereo although my Meiji EMZ1 lacks a trino' port and, like many stereo light-bases, has poor uneven lighting for camera work. But was interested to see how it performed for NIR and the setup is shown below. The OpticStar camera was used because of its live view and sensitivity. The under driven quartz halogen lamp was apparently emitting more NIR than visible as a neutral density filter had to be added for NIR work to stop overloading the camera.

Left above: Meiji EMZ1 stereo with the 6V 7W quartz halogen lamp base. The OpticStar camera sat in an eyepiece tube with 'C' mount adaptor or for wider field a Moticam relay lens on the Meiji W10X eyepiece. The Meiji supplementary 0.5x lens was added to the 1-3x zoom head. To improve the lighting, strong diffusers were added below the ground glass base plate.

Right above: Set-up used for NIR imaging with the 850 nm filter under slide (it's totally opaque to the eye). One slide edge sits on a strip of 6 mm thick mouse mat to present the slide at approx. the correct angle to one train of the Greenough stereo optics to give a flatter imaging plane.



Seven spotted ladybird, unnamed Victorian whole mount. The exoskeleton on this subject was very dense.
Left above: Brightfield with NIR blocking filter. Little internal detail was seen.
Right above: 850 nm long pass filter. Good internal detail was seen. 


Images below: as above, optical zoom in to show abdomen detail. Left - visible light, right - NIR.

There is of course a loss of resolution using NIR light but for the gross insect detail it was typically useful for it wasn't noticeable, except for whole insect imaging on the low NA stereo objective with 0.5x supplementary lens where the images did lack bite.

Comments to the author David Walker are welcomed.

Acknowledgements:
Thank you to Marien van Westen who offers his versatile Micam camera control, imaging and measurement software for free download and use. All image capture with the OpticStar above used Micam. I much prefer Marien's software to control the camera than the camera maker's supplied software.
The author's own casual interest in NIR microscopy was inspired by the notes of Tony Dutton in the Quekett Microscopical Club Bulletin (ref. 2). Hopefully my modest trials may also encourage others to have a go.
Thank you to the reader who suggested I tried blocking filters with a tungsten lamp, prompted by my earlier article using NIR LEDs. Unfortunately it was some years ago and can't remember their name, but will be pleased to acknowledge them if they'd kindly contact me.

References
1) R. P. Loveland, 'Photomicrography. A comprehensive treatise', Volume I, 1970. Figure 7-11, p.338, 'Spectral distribution of radiation from tungsten lamps'.
From this graph, a bulb run at 3000K has its peak wavelength in energy emission at ca. 800 nm i.e. in the near IR region. The Zeiss 12V 60W lamp when set at ca. 11.5V is at 3000K from a graph in Zeiss' Photomicroscope III manual 'G41 170  1-e', p.13.

Volume 2, chapter 14 ' Use of the whole Photographic Spectrum' of Loveland's book presents a valuable discussion of the application of infra red and ultraviolet microscopy in photomicrography with examples. The author notes the value of NIR for insect photography.

2) 'Constructing and adapting sub-miniature CCD TV cameras to the microscope' by J A Dutton. Bulletin of the Quekett Microscopical Club, 1997, 30, p. 21-22. Also the record of a demonstration by J A Dutton at the QMC Annual Exhibition (1997) on p.41-42 of the same issue.

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