Microvolume Spectrophotometry

Nucleic Acids and Proteins

1. Pre-Clean: With a MilliQ water squirt bottle, wet the tip of a low-lint lab tissue wipe (e.g. KimWipe) and wipe the Nanodrop/NanoQuant pedestal (base and top), the surfaces that contact the sample. Then wipe dry.
2. Initialize: Select the software program, e.g. Nucleic Acids or Protein
3. Blank: Load 1.2 µL of the solvent blank, e.g. water or Tris buffer for DNA.
   Click Blank on the software (F3). Wipe the pedestal dry.
   ^{1 µL is often used for DNA blank and sample. A little more can make it more precise and accurate. TECAN NanoQuant requires ≥2 µL per well, best with all wells used individually blanked, not averaged.}
4. Measure: Load 1.2 µL of your sample and click Measure (F1). Wipe dry. Repeat for remaining samples.
   ^{Use the same volume as the blank.}
5. Post-Clean: Wipe the pedestal again with the clean wet tissue and then with a clean dry portion, free of DNA.

Cell Culture

Microvolume: Pre-clean as before, but use the Cell Culture program with Pedestal option checked. Load no less than 2 µL of medium blank and cell culture. Make sure the culture is well-mixed before sampling.
To convert to the rough approximate equivalent 1 cm cuvette E. coli OD (at least for M9 media), multiply the microvolume OD₆₀₀ by a factor of 7.5–9. When estimated to be in the desired OD range, measure the OD in a cuvette.

Cuvette: In the Cell Culture program, ensure the Pedestal option is unchecked. Blank with 450–500 µL medium. Then measure 500 µL well-mixed sample culture in a separate cuvette. Alternatively, mix 50 µL culture into the 450 µL blank (or 55.5 µL into 500 µL) and multiply the measured OD by 10.
The cuvette should be gently shaken or tapped to further mix the culture.
An OD is only as reliable as the cell distribution uniformity of the source culture at the moment of sampling and the uniformity in the cuvette at the moment of measurement. Multiple readings may be averaged, with the cuvette shaken in between, necessary for cultures with aggregates/flocculants.

Measurement Linearity: OD readings are only in a reliable window of linearity with cell concentration when OD₆₀₀ = 0.1 – 1. When an OD is above this range or expected to be so, the culture must be diluted with the same medium before measuring, and the OD multiplied by the dilution factor. For example, 450 µL medium can be blanked, 150 µL culture mixed in to homogeneity, and then the OD reading multiplied by 4, as the reading is for 150/(450+150)=¼ the culture's concentration.

Note: The 2 mm sample beam passes through the cuvette 8.5 mm above the base of the cuvette.

Shyam's Sep 30, 2019 issue of Lab Improvement Weekly:

Some readers may be skeptical of using the Nanodrop microvolume pedestal for cell culture OD measurements.
I personally have always used cuvette measurements and thus require additionally culturing 2–3 × 700 µL extra volumes for measuring ODs in cuvettes in addition to what I need for an experiment that requires cells of a certain OD, e.g. mid-log phase. I found only 10–18% higher OD600 readings from the Nanodrop pedestal (2 µL) versus from cuvettes (700 µL) when the cultures were OD600=0.1–0.2 in M9CA. The conversion factor is pedestal OD × ~7.5–9 to get the equivalent cuvette measurement (including the 1 mm -> 1 cm pathlength conversion).
Cuvettes were loaded with pre-shaken cultures. Nanodrop samples were taken directly from measured cuvette samples, shaken to re-homogenize. 2 µL is necessary according to Nanodrop for reproducible microvolume cell density measurements. Thus, Nanodrop measurements are a good primary estimator for OD600. But once your cultures are estimated to be in the d esired OD range (often mid-log), you should still take a cuvette measurement if you need more accuracy, as when you need to dilute the culture to an exact OD. So still plan 500 µL for that measurement.

Minimum Cuvette Volume
Ever wonder how much liquid you need to make a minimal cuvette measurement? The manual says "Fill cuvettes with enough blanking or sample solution to cover instrument optical path (2 mm sample beam is 8.5 mm above cuvette bottom)."
I measure the bottom of the meniscus of 400 µL water in a standard cuvette to be 10 mm above the cuvette bottom, thus 1.5 mm above the optical path. So long as there aren't bubbles below the surface, 400–500 µL should be a reproducibly measurable volume and allows you to reduce the typically prescribed 1 mL measurement in half. I have switched my standard measurement volume to 500 µL <0.5 OD culture, or 450 µL blank + 50 µL high-OD culture mixed in.

DNA concentration measurement precision between TECAN NanoQuant plate and Thermo NanoDrop, in ng/µL:

Red Fluorescent Protein interference with OD measurement

"Red fluorescent proteins (RFPs) can strongly absorb light at 600 nm. Increasing RFP expression can falsely inflate apparent cell density and lead to underestimations of mean per-cell fluorescence by up to 10%. Measuring optical density at 700 nm would allow estimation of cell abundance unaffected by the presence of nearly all fluorescent proteins."

Hecht et al. (2016) When Wavelengths Collide: Bias in Cell Abundance Measurements Due to Expressed Fluorescent Proteins. doi.org/10.1021/acssynbio.6b00072

OD₇₀₀ is a suitable standard for microplate reader measurements, where cuvette-equivalent 600 nm OD is irrelevant. OD₆₀₀ should still be used for traditional protocols instructing a culture to be monitored and harvested at a particular OD, e.g. competent cells at mid-log, OD₆₀₀≈0.3.