Setups for High-Speed Multi-color Fluorescence Imaging

Thinking Outside the Filter Cube

Guest Authors: Brad Reynolds, Isabel Goodhand PhD., CoolLED 

When choosing filter sets for your experiments, it’s not just the optical properties of your fluorophores you need to consider. To enhance speed and contrast, the latest microscope technology has also introduced options in terms of where to position these filters – and this is no longer limited to the filter cube. 

We will be exploring a few different options for combining the latest technology in widefield fluorescence, comparing various filter types and their pros and cons.  

First and foremost, it’s important to clarify what we mean when discussing different filter configurations. Table 1 introduces different filter configurations. This example represents the excitation filters, dichroic mirrors, and emission filters for a 3-color experiment that requires UV, blue, and green excitation. The color of each filter represents the wavelength of transmitted light; solid colors represent single-band filters; split colors represent multi-band filters.  

Filter configurations are a trade-off in terms of speed and contrast.

Single-band filters provide the highest contrast, but for multi-channel imaging, a whole filter cube housed in a filter wheel can be slow. On the other end of the spectrum, full multi-band filter sets remove latency from mechanical movement. Mechanical movement slows channel switching speeds, especially when waiting for vibration to settle, and so bypassing this increases temporal resolution and the details that can be gleaned from live cell imaging experiments. The downside to multi-band filter sets is the risk of bleed-through which can compromise contrast. Pinkel and Sedat configurations sit in the middle of the speed vs. contrast trade-off and are the most interesting options when looking for a solution to achieve the best of both worlds. 

LEDs - the go-to illumination technology for widefield fluorescence 

This is where the light source comes into its own. Over the last decade, LEDs have become the go-to illumination technology for widefield fluorescence, replacing traditional mercury and metal halide lamps due to enhanced performance, convenience, and environmental credentials. For example, LED illumination systems remain stable over time, delivering precise experimental results. Moreover, electronic control options such as TTL triggering enhance temporal resolution to capture the most dynamic events during live cell imaging. Some models further enable high-speed imaging with the facility to house single-band excitation filters within the light source. Sitting in front of each LED channel, these allow a Pinkel or Sedat configuration, and in a similar way to full multi-band configurations avoid any latency of mechanical movement in the excitation light path.

Pinkel and Sedat Considerations

A Pinkel set can still present a compromise in fluorophore specificity due to the multi-band emission filter in the emission path, and the Sedat configuration is preferable if specificity is a priority. In these scenarios, the addition of an image splitter in the emission light path enables the use of a Sedat filter set while still avoiding mechanical movement – but this requires a higher optical power budget. 

Multi-Sedat Hybrid Sets Considerations

Since not all imaging experiments have the same aims, filter sets that allow another layer of flexibility can be useful in the lab. The Multi-Sedat hybrid set is ideal for switching between high-speed and greater specificity requirements, where a multi-band excitation and emission filter sit in the filter cube for high-speed applications. For experiments requiring extra specificity, single-band emission filters can be housed in a filter wheel and switched into the light path as needed.   

The Future of High-Speed Multi-Color Imaging

In the ever-evolving world of microscopy and fluorescence imaging, the choice of filter configurations and illumination technology plays a pivotal role in achieving the desired experimental results. As we have discussed, the landscape of filter setups has expanded, inviting us to think beyond the conventional filter cube. However, it is still important to consider the trade-off between speed and specificity.

Single-band filters excel in specificity, while multi-band filter sets provide speed by eliminating mechanical movements that can slow down imaging. However, they do carry the risk of bleed-through, which may compromise specificity. Pinkel and Sedat configurations represent a balance, offering both speed and specificity, making them particularly enticing for scientists seeking the best of both worlds. 

In conclusion, this is a perfect example of complementary technologies working together to enhance live cell imaging, and we always recommend thinking about the bigger picture of filter configurations when considering upgrading imaging equipment.   

Find out more about CoolLED and Chroma today:

• To learn more about the different LED Illumination Systems now available, visit www.coolled.com 

• Filter and Filter Sets optimized for use with LED Illumination Systems