# US 2026/0063531 A1 · In-line spectroscopic cytometry for real-time monitoring of celllular and/or molecular bioprocesses

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- Number: US 2026/0063531 A1
- Document ID: US20260063531A1
- Status: Published application
- Kind: A1
- Application number: 19/311,089
- Filed: 2025-08-27
- Published: 2026-03-05
- Earliest claimed: 2024-08-30
- Assignees: Physical Sciences, Inc.
- Applicants: physical Sciences, Inc.
- Inventors: Jesung Park; Youbo Zhao; William J. Kessler
- Web page: https://patentgrep.com/patent/US20260063531A1

## CPC

- G01N 15/1431: {the electronics being integrated with the analyser, e.g. hand-held devices for on-site investigation}
- C12M 41/46: {of cellular or enzymatic activity or functionality, e.g. cell viability}
- G01N 15/01: specially adapted for biological cells, e.g. blood cells (investigating sedimentation of particle suspensions in blood G01N15/05)
- G01N 15/1427: {with the synchronisation of components, a time gate for operation of components, or suppression of particle coincidences}
- G01N 15/1429: Signal processing
- G01N 15/1434: Optical arrangements
- G01N 15/1459: {the analysis being performed on a sample stream}
- G01N 33/5005: {involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025)}
- G01N 2015/1006: {for cytology}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}

## Abstract

An analytical method and system for monitoring cellular status wherein excitation energy is focused into an excitation cytometry volume within a cell sample that is located within a bioreactor or fermenter to induce optical signals from intracellular compounds. The resulting optical signals are directed to a detection subsystem that has at least two detection channels. One channel detects the elastic (Rayleigh or Mie) scattering signal from the cell that identifies the presence of the cell within the excitation volume. Another channel detects the fluorescence and/or Raman scattering signal of intracellular compounds of the cells. The elastic scattering signals associated with individual cells are used as event-triggers to gate the detection of the intracellular fluorescence and/or Raman signal to eliminate the background noises from the suspension media. The intracellular fluorescence and/or Raman scattering signal are used to characterize cellular status based on the fluorescence intensities at specific fluorescence wavelengths and peak analysis of Raman scattering spectra.

## Claims

### Claim 1

An in-situ cytometry method of monitoring cell status and reducing background fluorescence and/or background scattering signals from non-cellular material, the method comprising:
directing electromagnetic energy into a cytometry volume, located within an agitated bioreactor or fermenter, said cytometry volume being subcellular in size, to stimulate elastic scattering photons and fluorescence and/or Raman photons from a cell present in the cytometry volume;
directing elastic scattering photons from the agitated bioreactor or fermenter to a detector which outputs a trigger signal;
directing fluorescence and/or Raman photons from the agitated bioreactor or fermenter to a spectroanalysis subsystem; and
selectively spectroanalyzing the fluorescence and/or Raman photons in response to the trigger signal output by the detector to determine a biochemical profile of the cell and to reduce background noise.

### Claim 2 (depends on 1)

The method of claim 1 in which selectively spectroanalyzing the fluorescence and/or Raman photons includes not allowing the fluorescence and/or Raman photons to reach the spectroanalysis subsystem until the trigger signal is output by the detector.

### Claim 3 (depends on 1)

The method of claim 1 in which the spectroanalysis subsystem includes a spectrometer and a processing subsystem responsive to an output of the spectrometer, the processing subsystem triggered by the trigger signal to spectroanalyze the fluorescence and/or Raman photons.

### Claim 4 (depends on 1)

The method of claim 1 in which the fluorescence photons are from NAD(P)H and FAD intracellular metabolites.

### Claim 5 (depends on 4)

The method of claim 4 further including differentiating between NAD(P)H and FAD intracellular metabolites.

### Claim 6 (depends on 5)

The method of claim 5 further including calculating a fluorescence ratio FAD/(FAD+NAD(P)H) to quantify a fluorescence redox ratio.

### Claim 7 (depends on 6)

The method of claim 6 further including quantifying the redox ratio using a spectral band pass algorithm and/or a skewed Gaussian fitting algorithm.

### Claim 8

An in-situ cytometry system for monitoring cell status and reducing background fluorescence and/or background scattering signals from non-cellular material, the system comprising:
a probe insertable into an agitated bioreactor or fermenter, the probe defining a subcellular cytometry volume in the bioreactor or fermenter;
a source outputting electromagnetic energy to the probe to stimulate elastic scattering photons and fluorescence and/or Raman photons of a cell present in the cytometry volume;
a first detection channel directing elastic scattering photons from the probe to a detector which outputs a trigger signal;
a second detection channel directing fluorescence and/or Raman photons from the probe to a spectrometer; and
means for selectively spectroanalyzing the fluorescence and/or Raman photons in response to the trigger signal from the detector to determine a biochemical profile of the cell and to reduce background noise.

### Claim 9 (depends on 8)

The system of claim 8 in which the means for selectively spectroanalyzing includes a shutter configured to allow the fluorescence and/or Raman photons to reach a spectroanalysis subsystem in response to the trigger signal.

### Claim 10 (depends on 9)

The system of claim 9 in which the shutter includes a DMD.

### Claim 11 (depends on 8)

The system of claim 8 in which the means for selectively spectroanalyzing includes a processing subsystem responsive to an output of a spectrometer, the processing subsystem triggered by the trigger signal to determine a biochemical profile of the cell.

### Claim 12 (depends on 8)

The system of claim 8 further including a processing subsystem configured to determine a fluorescence redox ratio of the cell from the fluorescence photons.

### Claim 13 (depends on 12)

The system of claim 12 in which the fluorescence scattering photons are from NAD(P)H and FAD intracellular metabolites.

### Claim 14 (depends on 13)

The system of claim 13 in which the processing subsystem is configured to differentiate between NAD(P)H and FAD intracellular metabolites.

### Claim 15 (depends on 14)

The system of claim 14 in which the processing subsystem is configured to calculate a fluorescence ratio FAD/(FAD+NAD(P)H) to quantify a fluorescence redox ratio.

### Claim 16 (depends on 15)

The system of claim 15 in which the processing subsystem is configured to quantify the redox ratio using a spectral band pass algorithm and/or a skewed Gaussian fitting algorithm.