NanoDrop ND-3300 Overview

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NanoDrop 3300 Fluorospectrometer

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The Thermo Scientific NanoDrop^TM 3300 Fluorospectrometer is a multi-source optical instrument for measuring fluorescence of 1 µl samples . This space-saving fluorometer offers exclusive features that allow investigators the flexibility to use fluorometry in valuable new ways.

The NanoDrop 3300 performs broad spectrum fluorescent analysis with patent-pending technology that delivers a wide excitation range without requiring filter changes or a monochromator. The micro-volume capability lowers the fluorescent detection limit for sample mass by more than an order of magnitude over conventional fluorometers (e.g. two pg dsDNA – the equivalent of that in a single cell). In addition, it has operational simplicity comparable to less versatile instruments, making it easier to use for those users who have limited experience with fluorescence techniques.

The uniquely clean optics of the patented retention system, combined with virtual filtering (see below) for the white LED applications, enables measurements across a wide range of wavelengths using sample volumes of 1-2 ul without cuvettes and costly filter changes.

The excitation source comes from one of the three solid-state LEDs housed in a block adjacent to the measurement pedestal (see below for more details about the LEDs). Emitted light is collected using the traditional 90° angle from the respective excitation source. A 2048-element CCD array detector, covering 400-750nm, is connected by an optical fiber to the optical measurement surface. The spectrometer is configured with a cut filter to eliminate light transmission below 395 nm.

An added convenience is that no external power supply is required. All of the operating power is supplied by the USB port on the PC.

Excitation Sources and Applications

Excitation	Emission/Applications
UV LED max=365 nm; equipped with cut filter that eliminates excitation above 400nm	• GFP wt Em509 • Quinine Sulfate Em450 • Hoechst 33258 Em450 • 4-MU Em450 • Q Dots various
Blue LED max= 480 nm; equipped with cut filter that eliminates excitation above 495 nm	• GFP wt Em509 • EGFP Em509 • FITC-FAM Em515 • Alexa 488 Em520 • PicoGreen Em525 • RiboGreen Em525 • Alexa 555 Em565 • B-Phycoerythrin Em575 • Q Dots various
White LED range= 500-650; uses virtual filtering (see below)	• Cy3, Alexa 555 Em565 • Alexa 568 Em600 • Cy5, Alexa 647 Em667 • Sulforhodamine 101 Em 600 • 5-CMTREm570 • Q Dots various • TET Em535 • HEX Em555

Virtual Filtering

The white LED is composed of a blue LED and a yellow-orange phosphor excited by the blue LED to give an approximately white spectrum (typically bluish in appearance). Virtual filtering (patents pending) involves taking a reference spectrum as a wavelength intensity map of the white LED yellow peak. The reference spectrum is then normalized to a non-fluorescence wavelength of the sample's signal spectrum. This normalized reference spectrum, when subtracted from the signal spectrum, will remove nearly all the stray excitation contribution to the fluorescence signal spectrum. The normalization or scaling is typically done at a wavelength within the stability envelope (the region of the white spectrum) on the short wavelength side of the sample's fluorescence peak where the fluorescence signal is less than 5% of its peak value. This is illustrated in the figure below:


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