Macroscale Fluorescence Imaging

Fluorescence time-resolved image of NAD(P)H in a tumor. The borders of the tumor are marked with a white line. (Source: OSA)

Researchers have scaled up a powerful fluores­cence imaging technique used to study biolo­gical processes on the cellular level. Previously limited to samples just milli­meters in area, the expanded approach can analyze samples with areas up to 4 square centi­meters. With further develop­ment the new approach could find use in the clinic as a sensitive and precise method for identifying the edges of tumors during surgery.

The new macro­scale imaging approach is based on a biolo­gical imaging technique known as fluores­cence lifetime imaging micro­scopy (FLIM). “Our macro-FLIM system can not only obtain a sample’s structural infor­mation, but also allows observation of certain bio­chemical processes taking place within the sample,” said senior research scientist Vladislav Shcheslavskiy, Becker & Hickl, GmbH, Germany. “Although our goal is to develop this for clinical use, it could also be very useful in funda­mental studies for probing bio­logical processes as disease develops or inves­tigating biolo­gical responses to dif­ferent types of therapy.”

Now, the researchers demon­strate the first confocal micro­scopy based macro-FLIM system with cellular reso­lution and high molecular sensi­tivity. They used it to observe the metabolic processes inside a whole tumor in a live mouse, a feat that isn’t possible with current FLIM systems. The new system was produced through a close colla­boration between engineers and physicists from Becker & Hickl GmbH, Germany, and biologists from Pri­volzhskiy Research Medical Univer­sity, Russia. In addition to biolo­gical and clinical samples, the new macro-FLIM system could be used to analyze other samples with large areas. For example, it could offer a non-destructive method for deter­mining the media used in paintings that need restoring.

FLIM involves precise measure­ments of the fluores­cence decay rate, or lifetime, of a naturally fluores­cent molecule or a fluores­cent tag that has been added to tissue. Because the lifetime depends on charac­teristics of a molecule’s environment such as tempera­ture and pH as well as on its inter­action with other surrounding molecules, FLIM can be used to obtain infor­mation about the properties of the molecule and its micro-environ­ment.

Typi­cally, FLIM is performed using laser scanning confocal micro­scopy, which achieves high resolution by scanning a laser beam across a fluorescent sample to form an image. To obtain FLIM infor­mation on a macro scale, the researchers developed a confocal macroscopy system that incor­porates lasers with extremely short pulses only pico­seconds in length and very sensitive detectors to sense the fluores­cence. The system also included elec­tronics that count the photons and plot their distri­bution in relation to the time since the laser pulse and the position of the laser beam on the sample.

Shcheslavskiy continued: “Careful optical design along with the pico­second lasers, sensitive and fast detectors and fast single photon counting elec­tronics allowed us to record fluores­cence decay with high precision at macro­scale.” Confocal micro­scopy is typically limited to an imaging area of just millimeters but placing the samples in the macro­scanner’s inter­mediate image plane allowed the researchers to image larger samples. They then plotted the photon distri­butions across a large area of the sample to acquire macro-scale fluores­cence lifetime infor­mation.

To demon­strate the cellular reso­lution of the macro-FLIM system, the researchers used it to image fluores­cent micro­beads with a diameter of 14.6 microns and live cultured cancer cells labeled with fluores­cent dye. They then used the macro-FLIM system to analyze an entire tumor in a live mouse. They did this by simul­taneously measuring the fluores­cence lifetime of a gene­tically encoded red fluores­cent protein, which identi­fied the location of the tumor, and nicotinamid adenine dinu­cleotide (NADH), a molecule respon­sible for energy production in living cells.

“The sensi­tivity of our system was high enough to observe fluores­cence of intrinsic tissue components such as NADH without any labeling,” said Shcheslavskiy. “In addition to being used to study meta­bolism in a tumor, macro-FLIM could be used to follow cell death or oxygen status of tumors on a macro­scale with cellular reso­lution.” To develop the system for clinical appli­cations, the researchers are working to improve its flexi­bility and mobi­lity. They also want to combine the macro-FLIM system with a scanning stage that would move the sample to allow FLIM to be performed on areas as large as 10 by 10 centi­meters. (Source: OSA)

Reference: V. I. Shcheslavskiy et al.: Fluorescence time-resolved macroimaging, Opt. Lett. 43, 3152 (2018); DOI: 10.1364/OL.43.003152

Links: Becker & Hickl GmbH, Berlin, Germany • Institute of Biomedical Technologies, Privolzhsky Research Medical University, Nizhny Novgorod, Russia

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