Custom Microscopy Illumination

What’s New in Custom Microscopy Illumination?

The latest custom microscopy illumination technology can offer many benefits when building a microscope-based system for applications such as high-content screening or slide scanning.

Illumination can be a complex topic and many variables must be considered – not least spectral coverage, irradiance and speed. First and foremost, you also need to decide between metal halide, lasers and LEDs. LED illumination technology continues to evolve at a rapid pace, overtaking the performance of metal halide lamps, and in some cases even offering a viable alternative to lasers.

Following several breakthrough advances, LEDs are now often considered the go-to option for microscopy illumination.

CoolLED and Custom Microscopy Illumination

CoolLED is a global leader in designing and manufacturing cutting edge LED custom microscopy illumination using the latest LED technology.

Since we introduced the first commercially available LED Illumination System for fluorescence microscopy in 2006, we have led the way in transforming fluorescence and transmitted illumination. We now offer a diverse off-the-shelf product range, from 8-channel systems to those specialised for ratiometric calcium imaging – and everything in between! For those of you who need something a little different, we also draw from our well-established mix of science and engineering know-how to offer custom microscopy illumination: take a look at our technology.

Unprecedented Choice of Wavelengths

LED availability is growing all the time. Only a few wavelength options existed in 2006, and now we can cover 340 – 850 nm, from the UV right through to the IR. Anywhere from two to eight discrete LED channels are also possible, opening up a host of multi-label imaging applications.

Successful fluorescence microscopy also depends on matching optical filters with the custom microscopy illumination and the fluorophore(s) of choice. It is important to ensure that optimal filters have been carefully selected for the experimental set up, and to be aware that many standard filter sets have been optimised for mercury and metal halide peaks rather than for LEDs, which can compromise image quality.

To overcome this, we offer recommendations for filters perfectly matched to the spectra of CoolLED LEDs and common fluorophores. We can also work alongside you to determine the best optical filters for your requirements: find out more about working with us.

Brighter Custom Microscopy Illumination

When LED illumination first arrived on the market, light output struggled to match metal halide systems. But CoolLED’s R&D team are always innovating, and our LEDs now surpass the irradiance of metal halide systems in many areas of the spectrum. In fact, our LEDs are often used at levels far below their 100% irradiance limit.

When it comes to measuring light output in custom microscopy illumination, irradiance is an important term to remember and overcomes the many confusing statements you may encounter around power, intensity and radiant intensity. Irradiance describes the radiant power at a surface per unit area, measured in mW/mm². This is our preferred term as measured at the sample plane – the only place that counts. You can read more about this topic in our white paper.

It is also important to remember that higher irradiance is not always beneficial, and lower levels protect against photobleaching and phototoxicity. This is where control of the custom microscopy illumination comes in, matching high maximum irradiance with the ability to optimise it for individual applications.

Controlling Custom Microscopy Illumination

The ease and efficiency of LED control sets them apart from traditional lamps for custom microscopy illumination. Each individual channel can be controlled with on/off operation, or the irradiance tuned between 0-100%. Alternatively, a selection of channels or all channels in the system can be controlled in the same way as one, for example to provide white light illumination.

This could be achieved via USB (Windows, GUI and serial command language) where applications require fast, but not ultra-fast imaging. Here, speeds can vary depending on the individual PC setup and software overheads.

For applications requiring ultra-fast imaging, whether for high-speed slide scanning or capturing fast events with high temporal resolution, hardware synchronisation is available to bypass the limitations of computer latency. This transistor-transistor logic (TTL) offers the fastest acquisition possible by electronically synchronising the hardware. For example, by connecting the camera TTL-out to the LED custom microscopy illumination, this will trigger the desired LED (or selection of LEDs) during camera exposure.

This approach can achieve speeds as fast as 20 µs, and analogue offers high-speed switching and irradiance control (0-10 V, 0-50 kHz).

Taking hardware synchronisation one step further for multi-label imaging, inline excitation filter holders allow a Pinkel optical filter configuration (with single-band excitation filters but a multi-band dichroic mirror and emission filters). This approach to custom microscopy illumination achieves high contrast imaging, while capitalising on the TTL switching speeds of the LED, as it removes the latencies associated with filter wheels (see below).

Removing these extra moving parts also makes the overall illumination system far more robust and reliable over time, while photodamage (photobleaching and phototoxicity) are reduced when limiting illumination only to image acquisition.

If you’re interested in seeing how CoolLED custom microscopy illumination can enhance your system build, please contact us at [email protected] for more information.