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NIR Lasers Power Advanced Multiplexed Confocal Imaging

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Sample images taken with an Olympus confocal microscope

Confocal laser scanning microscopes are popular biological research tools. They're commonly used to image multiple fluorophores at once with good color separation, as well as image deep within a biological specimen with enhanced sectioning capabilities.

Today, the latest laser tech innovations can benefit applications like these and enable more advanced experiments. In this blog post, we’ll discuss how new near-infrared (NIR) excitation lasers on our FV3000 confocal microscopes are powering advanced multiplexing applications.*

Obstacles to Multiplexing with 5+ Channels in Confocal Microscopy and How to Overcome Them

Let’s start by casting light on the history of multiplexing experiments.

For years, many researchers performed immunofluorescence with DAPI and two other colors, typically in the green and red spectra.

As antibodies and imaging systems advanced with more detectors and better emission wavelength filtering, four-color immunofluorescence became popular. DAPI, green, red, and far-red colors were the most common four-color combination.

Yet, two critical factors created obstacles toward the introduction of a fifth channel for multiplexing.

1. A lack of NIR laser diodes with good beam qualities.

First, NIR laser diodes with good beam qualities for confocal laser scanning microscopes were not widely available. Adequate power (but not too much power), minimal power fluctuations, and compatible beam profiles are all necessary features of laser diodes used in confocal imaging. Yet, only a few NIR laser diode options were available in these wavelength ranges until the past few years.

But this has all changed thanks to the latest laser diode technology. Our FV3000 confocal microscope now offers 730 nm and 785 nm laser diodes for efficient excitation of secondary dyes, such as:

  • Cyanine7 (Cy7)
  • Alexa Fluor 750
  • Alexa Fluor 790
  • DyLight 800
  • IRDye 800
  • Indocyanine green (ICG)

These dyes, along with a growing number of new fluorophores, are making the addition of a fifth and sixth simultaneous channel for multiplexing more attractive.

2. Photomultiplier tubes with reduced sensitivity in NIR wavelengths.

A second obstacle is that many photomultiplier tubes (PMTs) have reduced sensitivity in the detection wavelengths typical for 730 nm and 785 nm excitation.

This reduced sensitivity in the near-infrared detection ranges is especially true for popular GaAsP PMTs that offer higher sensitivity in the middle of the visible spectrum. In the 750+ nm range, GaAsP detectors have very little sensitivity.

To overcome this challenge, we integrated red-shifted GaAs detectors into our FV3000 confocal laser microscope.

Our FV3000 system now offers GaAs detectors in many different combinations with multialkali (standard) PMTs and GaAsP (high sensitivity) PMTs. These configurations can use GaAsP detectors as the fifth or sixth channel* for multiplexing applications. (*Some limitations apply)

Combined with our X Line high-performance objectives, our FV3000 system can provide high-quality broad chromatic aberration correction from 400–1000 nm. This results in much better color reproducibility during brightfield and multicolor fluorescence imaging.

*This is a customized solution and may not be available in some regions. Please contact your local Olympus representative to get detailed information.

Related Content

Brochure: FV3000 Confocal Laser Scanning Microscope

Video: FV3000 Confocal Microscope Product Introduction

Resource Center: Customized Solutions for Tailored Discovery

Manager Life Science Applications Group

James Lopez received his Ph.D. in biomedical sciences from the University of Chicago in 2010. With nearly a decade of experience in calcium imaging, FRET, live cell imaging, and intravital imaging, James joined Olympus as a confocal and multiphoton sales representative. He later transitioned to the Olympus Life Science Applications Group supporting confocal and multiphoton systems. Now he manages the Life Science Applications Group in the US, Canada, and Latin America markets.

Jan 21 2020
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